Jarrell Miller has returned a second "adverse finding" from a drugs test and will now be replaced as Anthony Joshua's next opponent, says Joshua's promoter Eddie Hearn.
Miller was denied a licence for the 1 June bout after a first adverse finding from a sample taken on 20 March.
Hearn said Miller had "failed a second separate test for a further substance".
He says a new opponent for British IBF, WBA and WBO heavyweight champion Joshua will be announced next week.
The fight, due to take place in New York, will be Joshua's US debut.
Hearn had previously stated that Joshua's "preparations continue" and that eight boxers were being considered as Miller's replacement.
After the American returned the first adverse sample on 20 March, his team requested that a B sample be tested.
Before the report of the second adverse finding, the 30-year-old said that he had "done nothing wrong".
"The facts will prevail and I shall be vindicated," Miller said on Thursday.
"My team and I stand for integrity, decency and honesty and we will fight this with everything we have."
Hearn said he had been informed of Miller's second adverse finding by the the Voluntary Anti-Doping Association, which operates within boxing and mixed martial arts.
Among those under consideration to replace Miller are Cuba's Luis Ortiz, Poland's Adam Kownacki and American Michael Hunter.
"It feels like we need an American fighter or someone that the American market is also familiar with," Hearn previously told BBC Sport.
"Ultimately we want someone from the top 15 that's going to put up a great fight, who is going to come to win. That's the most important thing. I don't want someone who wants to take a payday on 1 June, I want someone who wants to come and rip the world heavyweight title from Joshua."
Joshua, 29, is undefeated in 22 bouts and unified three of the heavyweight division's world titles by beating Wladimir Klitschko in the summer of 2017.