Native River has been ruled out of the Magners Cheltenham Gold Cup with a suspensory tendon injury.
Winner of the race in 2018, he would have been heading back to the Festival next month aiming to become the first horse since Kauto Star in 2009 to regain the blue riband event.
The 10-year-old had appeared right back to his best this season, winning at Aintree and only recently in the Denman Chase at Newbury.
He was also prominent in the betting for the Randox Health Grand National, although his participation in that race had still to be confirmed.
Trainer Colin Tizzard's son and assistant Joe said: "Unfortunately we've had to scratch Native River from the Gold Cup this morning - he's picked up a suspensory tendon injury, which means his season is over."
The Tizzards do still have a valid Gold Cup contender in their yard, with Betfair Chase winner Lostintranslation.
Native River was in line to appear at his sixth successive Cheltenham Festival.
He was unplaced in the Albert Bartlett Novices' Hurdle in 2015 but has been in the frame ever since.
The following year he chased home Minella Rocco in the National Hunt Chase, before finishing third to Sizing John in the 2017 Gold Cup.
He then came out on top a year later in a thrilling battle with Might Bite to take the most sought-after prize in steeplechasing - and despite connections intimating he was never at his best last season, he still finished fourth in defence of his crown.
Native River had been around the 10-1 mark in the betting - and as a result of his withdrawal, those at the head of the betting were trimmed slightly.
