A much-changed Scotland missed out on the chance to gain Nations League promotion after losing 1-0 to Slovakia.
Jan Gregus's deflected 31st-minute strike proved the difference between the two sides in Trnava, although Steve Clarke's team still have another opportunity to win Group B2.
If Scotland win in Israel on Wednesday night, they will be assured of promotion to the Nations League top tier, although that will also be confirmed if Israel and the Czech Republic play out a draw in their clash on Sunday night.
Player ratings
Slovakia: Rodak (8), Pekarik (6), Satka (6), Skrinair (7), Mazan (6), Hrosovsky (6), Duris (6), Kucka (6), Hamsik (7), Gregus (7), Duda (7)
Subs: Rusnak (6), Safranko (6), Lobotka (6)
Scotland: Gordon (7), Cooper (7), McKenna (6), Considine (6), Palmer (8), McGinn (7), McLean (7), Armstrong (7) Tierney (9), Christie (8), McBurnie (6)
Subs: Shankland (6), Griffiths (7)
Man of the match: Kieran Tierney
How Scotland's unbeaten run was ended
The visitors arrived at the Stadion Antona Malatinsky buoyed by Thursday night's dramatic penalty shoot-out win over Serbia that earned them a place at next summer's European championship, although the exertions from that game meant Clarke rung the changes.
The Scotland manager made eight alterations to his line-up, with only Kieran Tierney, John McGinn - who was made captain - and Ryan Christie keeping their places, not that you would have known.
The Scots dominated for large chunks of the contest, only to fall behind against the run of play when Gregus's deflected shot from the edge of the box found its way past a rooted Craig Gordon and into the bottom left-hand corner of the net.
Team news
Slovakia made four alterations from their play-off final with over Northern Ireland with Robert Mazan coming in at left back, Patrik Hrosovsky taking on the defensive midfielder role, Jan Gregus starting and match-winner Michal Duris getting the nod in attack.
Meanwhile, the visitors made eight changes, with Kieran Tierney, John McGinn and Ryan Christie the only players who started Thursday’s Euro 2020 play-off final victory in Belgrade.
Craig Gordon, Scott McKenna, Liam Cooper, Andy Considine, Liam Palmer, Kenny McLean, Stuart Armstrong and Oli McBurnie all came in.
Clarke's side kept pushing for an equaliser after the break, with Kenny McLean going closest, only for his header to be kept out by Marek Rodak's impressive reaction save.
Slovakia were always a threat on the break as Scotland pushed more men forward, although it was the visitors who came closest to getting the next goal when in the final minute of stoppage time, Leigh Griffiths found himself with a sight of goal.
However, the substitute's goal-bound strike was brilliantly turned behind by Rodak's outstretched hand as Scotland's nine-game unbeaten run came to an end.
The end of Scotland's unbeaten run - Opta stats
- Scotland have lost just their second UEFA Nations League match (previously P8 W6 D1 L1), failing to score for the first time in the competition.
- Each of the last three matches between Scotland and Slovakia have finished 1-0, with this the first of those three matches to see Slovakia victorious.
- This was Scotland's first defeat since October 2019 against Russia, ending a nine-game unbeaten run (W6 D3).
- Slovakia have won their first home international match in a year, last winning such a match against Azerbaijan in November 2019.
- Scotland forward Ryan Christie was fouled seven times in this match, the most fouls a Scotland player has won in a UEFA Nations League tie.
- Slovakia midfielder Ján Greguš has scored four goals for Slovakia with each coming in different calendar years, starting in 2017.
- Scotland goalkeeper Craig Gordon has gone five matches without a clean sheet for the national team.
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What the manager said...
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Steve Clarke felt Scotland didn't get the result their performance deserved as they lost 1-0 to Slovakia in the Nations League but he does feel they are still progressing.
Man of the Match - Kieran Tierney
The left back was a constant threat to Slovakia all afternoon long with his lung-busting runs down the left flank, so much so that the only way the home team could stop the 23-year-old was to foul him.
The Arsenal left back's centres were particularly dangerous and none more so than when he picked out Kenny McLean in the second half, only for Marek Rodak to keep out the Norwich midfielder's header.
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What's next?
Slovakia travel to the Czech Republic, while Scotland visit Israel, with both Nations League games taking place on Wednesday at 7.45pm.