Best Mate will miss next week's Cheltenham Gold Cup after breaking a blood vessel on the gallops this morning.
The 10-year-old was bidding for a fourth successive win in the Grade One event next Friday but trainer Henrietta Knight has confirmed he will not run again this season.
She said: "He has broken a blood vessel on the gallops this morning and he will not be running at Cheltenham."
"Whenever they break a blood vessel it is serious but it is not career-threatening and hopefully he will come back as good as new next season," said Miss Knight.
"It will not have healed in time for the Gold Cup, it is very disappointing."
Best Mate was the general 5-2 favourite for the race despite being beaten on his last start at Leopardstown over Christmas.
The Jim Lewis-owned gelding was a distant second to Beef Or Salmon but he was found to be coughing after the race.
Knight feels that virus may be responsible for Best Mate's current problem.
She said: "These things are often caused by a virus and he had picked one up while he was in Ireland and I think it is all linked. He will have a long rest now though and will not run again this season."
Best Mate became the first horse since Arkle in the 1960s to take three Gold Cup when he was successful at the Festival last year.
Kingscliff is now the 7-2 favourite for race. Strong Flow is next best 5-1 with Irish horses Beef Or Salmon and Kicking King next best on 11-2.
Kicking King is set to run in the race, trainer Tom Taaffe has confirmed.
The seven-year-old was initially ruled out last week after failing to please the County Kildare handler in a workout and scoping badly.
However, a course of antibiotics seems to have done the trick and Taaffe reports the King George VI Chase winner to be back in the Cheltenham running.
In a statement, the trainer said: "Kicking King will be confirmed on Saturday as a runner in the totesport Cheltenham Gold Cup.
"The horse has surprised as to his well-being in all aspects since Sunday, March 6th and seems to have put his problems behind him. He has worked well and scoped well since and we hope all remains on course."