Staff at a bird sanctuary were shell-shocked when an eagle owl they had believed was male for 23 years unexpectedly laid an egg.
Kaln, who came to Gloucester's Barn Owl Centre as a chick in 1996, delivered its surprise on Sunday.
Founder Vincent Jones said: "You should have seen his face after he laid it. You should have seen our faces."
Kaln's sex had never been checked before as the bird was so healthy, Mr Jones said.
"The egg came as a massive surprise. He's now a tomboy," he said.
"His brother is still staring into the nest scrape in the ground confused to hell. He's now got a sister instead of a brother."
Mr Jones said although the egg was not fertilised and would not develop into a chick, there could be more as owls lay up to three or four at a time.
Staff planned to change Kaln's name to Kalnee in recognition of his newly discovered gender, Mr Jones said.
"We had no idea, because he always had the hallmarks of a male bird, and is a complete showman and loves people," he added.
Eagle owls can live for up to 60 years, and Mr Jones described Kaln as "still a bit of a teenager".
The bird has recently suffered health problems, including an inflamed heart, and the centre has launched an appeal to help pay for treatment.
"Hopefully it's a good indication if he's laid an egg that he's going to be OK," Mr Jones said.
The charity cares for 46 birds and specialises in helping rescued owls and birds of prey - both wild ones and those bred in captivity.