Cheltenham Gold Cup day began on a sombre note as favourite Sir Erec suffered a fatal injury in the opening race, the Triumph Hurdle.
The horse was pulled up by jockey Mark Walsh after breaking down midway through the race.
It was the second fatality of the meeting after the death of Ballyward in the National Hunt Chase on Tuesday.
Pentland Hills won the race with trainer Nicky Henderson saying: “What a gorgeous horse Sir Erec was.”
He added: “Your heart goes out to them.”
Winning jockey Nico de Boinville said: “You never like to win a race when that has happened. Thoughts are with Sir Erec’s connections.”
Retired 20-time champion jockey Sir AP McCoy, who is an adviser to McManus, told ITV Racing: “I’m heartbroken. It is one of the toughest things about the sport.”
A raft of new safety measures were brought in for this year’s festival after six horses died at the meeting 12 months ago. A seventh horse later died after suffering injuries at the fixture.
Racing’s rulers have been under pressure to improve safety from welfare groups and some politicians.
Sir Erec, trained in Ireland by Joseph O’Brien for owner JP McManus, was sent off favourite for the two-mile race for four-year-olds after winning a Grade One hurdle race at Leopardstown last month.
The horse, a son of Camelot – winner of the Derby at Epsom in 2012 – had earlier in his career been placed in a Group Two race on the flat for Joseph O’Brien’s father, Aidan.
Sir Erec, who was a hot favourite for the race with Joseph reporting that he had recovered from a sore foot last week, lost a front shoe before the race and the contest was delayed for several minutes as a farrier fitted a new one.
After the injury, Great British Racing chief executive Rod Street said on Twitter: “Oh my word. Standing there as such a gentlemen having his shoe replaced and looking beautiful. Then gone. Poor #SirErec What a low following yesterday’s highs.”
It was a very different mood to the previous day when Cheltenham was cheered by heart-warming wins for Frodon and Paisley Park.
As part of new measures this year, each of the runners – which number around 450 in total – have to pass veterinary checks before being allowed to race.
David Sykes, director of equine health and welfare at the British Horseracing Authority, told ITV: “There was no reason we could have predicted that injury.
“Beforehand when he was checked, the vets reported he moved well and had no indication of lameness or injuries.”
A statement from the course added: “As animal lovers, we hate to lose any horse and of course we shall review the incident.”