London Welsh are "extremely disappointed" to have been removed from the Greene King IPA Championship.
The storied London club fell into liquidation in last month, before being granted a temporary licence that expired on January 21.
A new board was challenged to provide evidence of funds to pay creditors and clarity on a new business plan but RFU bosses were left unconvinced.
In a statement, the club said: "London Welsh is extremely disappointed by the decision (on Tuesday) of the RFU to expel the club from the Greene King IPA Championship.
"The club has worked very closely with Nigel Melville, Ian Ritchie and Angus Bujalski of the RFU for a number of months to meet the criteria for a permanent licence, and the new company (Rugby 1885 Limited) believes that it did satisfy all of the conditions required in RFU regulation 5 and the insolvency protocol.
"The club will begin dealing with the ramifications of (Tuesday's) announcement before deciding whether to respond further."
An RFU statement, issued on Tuesday, read: "Following the appointment of liquidators in December 2016, a temporary licence was granted by the RFU to London Welsh, via a 'Phoenix Entity' to enable participation in the Greene King IPA Championship.
"The licence expired on January 21, 2017.
"The conditions required by the RFU regulations to enable London Welsh to continue to participate in the competition following the expiry of the temporary licence have not been met - despite the RFU granting two extensions to deadlines - and, with regret, London Welsh will no longer be granted permission to play in the Greene King IPA Championship for the remainder of this season and beyond."
Former Aviva Premiership outfit Welsh became one of the amateur domestic game's glamour clubs in the 1960s and 70s, boasting Wales stars like John Dawes, JPR Williams, Gerald Davies and John Taylor.
But the Richmond-based club have struggled to move with the times, and falling into liquidation in December has now proved fatal to their professional status.
The RFU, meanwhile, confirmed that Welsh's remaining Championship fixtures and points have been expunged.
"There will be no relegation from the Greene King IPA Championship this season, in line with RFU regulations," the RFU said.
"The Greene King IPA Championship will consist of 12 teams next season, including the team promoted by winning National League One."