A pack of Mackenzie River wolves, normally found in North America, had been a feature at the Highland Wildlife Park, Kincraig, near Aviemore, since 1972.
However, it has been revealed that the remaining six animals, aged between six and eight, have been destroyed because they were no longer displaying natural behaviour.
The Royal Zoological Society of Scotland, which owns the park as well as Edinburgh Zoo, said the deaths were carried out humanely and with the backing of the independent Animal Welfare and Ethics Committee.
Animals rights protesters have condemned the move, but the society says it was done after prolonged discussion and research.
A spokeswoman for the park said the catalyst for recent problems was that the pack's leader died of natural causes some time ago and the female did not accept the next male wolf in the pecking order.
"When things start to go wrong and they lose their pack structure they start very aggressive behaviour and start attacking each other when there is no natural leader," she said.
"That for us is where the welfare issue comes in. If they were in the wild it would be resolved quickly as they could move away from each other, but they are not and they would have continued to attack each other."
She said putting the animals down was considered to be more humane than leaving them to kill each other.
David Windmill, the society's chief executive, said: "Animal management is a complex, difficult, but rewarding work. With any kind of management, at times difficult decisions need to be taken and this was one of those times.
"The welfare of the animal is always paramount in our minds, and no decision is made until a full investigation has been carried out, taking into account all aspects of the species and the situation."
He added: "In the wild, animals are competing in the deadly game of survival of the fittest.
"Zoos have saved a number of these species from imminent extinction; the challenge will be to manage them in captivity to the best of our ability in the future, until perhaps one day the wild is safe enough for their return."
The pack, which was destroyed in January, has been replaced by Scandinavian wolves and the Kincraig park is now part of a European conservation breeding programme for vulnerable sub-species.
The wildlife park's Mackenzie River pack arrived in 1972 , but breeding of the animals ended in 2000.
Ross Minett, the director of Advocates for Animals, said the cull was "scandalous".
He added: "I don't think the general public is aware this kind of thing goes on in the name of conservation.
"This is the darker side of zoos' work, which I'm sure they don't want the public to find out about, and it typifies zoos' treatment of animals without paying them individual respect. They are seen to some degree as disposable commodities.
"Wolves kept in the kind of environment they are in in the wildlife park are unlikely to show the full repertoire of natural behaviour anyway, in a comparatively barren, deprived environment.
"We think this is pretty scandalous and believe the Royal Zoological Society has to answer how it can justify doing this.
"Can it continue treating in this manner animals supposedly under its care?"