The Chatsworth Horse Trials have been called off because of heavy rainfall.
Several potential Olympic combinations were due to take part, including Zara Phillips, William Fox-Pitt, Mary King, Piggy French and Nicola Wilson.
It follows the cancellation of the Badminton Horse Trials 10 days ago.
British hopefuls now have little time in which to practise before the Olympic team is announced on 24 May, though plans are being made to try to arrange another event prior to that date.
Organisers confirmed in a statement they had abandoned the Chatsworth trials, saying "even with good weather from now on, the footings would be unsafe".
An extra day of dressage had been added to the schedule to make up for the the cancellation of the Badminton trials.
Will Connell, the British Equestrian performance director, admitted the cancellation was a blow - but that there was no panic.
"We will now sit down with the selectors, riders and coaches and make a plan," he said.
"We will not rush into announcing this plan as we must also take a view on what the weather has in store and what options we have.
"In 2001, foot and mouth played havoc with the spring programme but we still prepared for - and delivered - at the European Championships.
"It is not the time to predict doom and gloom."