The Rugby Football Union has confirmed that Wasps - currently without a home - will play in next season's Championship.
But, having first rejected Worcester Warriors' application to participate on 16 December, the RFU has also now confirmed that they will not take part.
This followed discussions with Warriors' new owners, the Atlas Group.
Following their departure from Coventry after going into administration, Wasps remain without a ground to play at.
A Wasps spokesperson said: "We remain in positive dialogue with the RFU and thank them for their continued support as we work to secure our future.
"Recent events have highlighted, more than ever, the importance of long-term sustainable investment.
"Our focus remains on stabilising the club financially and Wasps playing in the Championship next season."
It has been speculated that Wasps themselves will move to Sixways next season - and Atlas co-owner Jim O'Toole had advised that an announcement on that was expected on Monday.
But Wasps have also been linked with playing at Damson Park, the home of National League football side Solihull Moors, and that is still at the negotiating stage.
The RFU confirmed Wasps' place after receiving the necessary financial assurances from their owners, HALO22 Limited, led by Chris Holland, who owns the club's Henley-in-Arden training ground and was a director with the previous Wasps regime.
They completed their purchase of Wasps on 16 December, the same day that the RFU first announced this same decision - two months ago.
But Atlas, who failed the owners' test in December, have again failed to satisfy the RFU's criteria - and they withdrew their application for a place in the division.
The RFU said they will "continue to work with Atlas over the coming weeks to understand their vision for rugby at Sixways".
But no mention has yet been made of whether the consortium bid,made by former Worcester director of rugby Steve Diamond and club sponsor Adam Hewitt, is still on the table.
Frustratingly for Warriors fans, Diamond told BBC Hereford & Worcester last week that they had passed the same tests as those failed by Atlas and could have guaranteed Championship rugby at Sixways next season.
But they were not the ones chosen as preferred bidders by administrators Begbies Traynor after the club went into administration in September.
More to follow.