Copenhagen Zoo euthanised two older animals and two cubs on Monday to make way for a new male.
The institution said: "Because of the pride of lions' natural structure and behaviour, the zoo has had to euthanise the two old lions and two young lions who were not old enough to fend for themselves."
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The new lion will be introduced to the two resident females who are both at breeding age.
Chief executive, Steffen Straede, said: "The zoo is recognised worldwide for our work with lions, and I am proud that one of the zoo's own brood now forms the centre of a new pride of lions
The zoo caused outrage last month when it killed Marius the giraffe, dissected his corpse in front of a crowd full of children and then fed the meat to lions.
Spokesman Tobias Stenbaek Bro said the European Association of Zoos and Aquaria recommended the zoo put down the giraffe because there were already a lot of giraffes with similar genes in the organisation's breeding programme.
He said EAZA membership is not mandatory but most responsible zoos are members of the organisation, which works to conserve global biodiversity.
Ben Williamson from PETA, said: "For anyone who cares about lions, giraffes and all the other individuals serving life sentences in zoos, the latest killings should confirm that zoos are hideous institutions that do not deserve public support.
"The way to protect "exotic" animals is to donate to campaigns that protect them in their native habitats."