Velazquez will ride Verrazano, a horse unbeaten in four races, the last of which came in the Wood Memorial.
Alongside him, Castellano will be in the saddle of Normandy Invasion, who came second behind Verrazano in the Wood Memorial.
Puerto Rico-born Velazquez, already a winner in the Derby on Animal Kingdom in 2011, will have a lot to do to get the better of Venezuelan Castellano, but he also faces a challenge from Joel Rosario.
Rosario will be partnered for the day by Orb, the horse normally favoured by Velazquez.
Rosario may fancy his chances, but in Velazquez and Castellano he faces two experienced jockeys, who have traditionally done well in Triple Crown events; Velazquez won the Belmont Stakes in 2007 and 2012, while Castellano was victorious at the 2006 Preakness Stakes.
In what will be the 139th Kentucky Derby, Velazquez is aiming to prove that at the age of 43 he still has what it takes to compete at the highest level, despite the injuries that he has sustained recently - he fractured a rib and cracked a bone in the seventh race at Aqueduct on April 8.
Thirty-six-year-old Castellano, meanwhile, has just one thing on his mind: to finally add a second Triple Crown title to his honours with the big one at Churchill Downs.