The RSPCA said the chickens are held in great affection
About 70 chickens have disappeared from a large brood which roosts on a roundabout in Suffolk.
The RPSCA is investigating after the flock at Ditchingham reduced from 100 birds at New Year to 30 at the beginning of July.
Chicken roundabout, as it is known, is at the junction of Norwich Road and the A143, and has been home to chickens and other poultry for at least 50 years.
The RSPCA said anyone found harming the birds would be prosecuted.
No bodies or injured birds have been found and it is not known whether there are any suspicious circumstances.
RSPCA Inspector Ben Kirby said: "Some may have been taken by foxes or other natural predators, but there is a possibility that others may have stolen or harmed.
"As we have no evidence, we must ask that anyone with information about anything suspicious gets in touch with us immediately.
"I would also remind people that they could be prosecuted if they are found to be harming the birds."
It is thought the birds originally escaped from allotments nearby and lived as a colony, surviving on grain from a nearby maltings.
They have come to be held in great affection by many local people, the RSPCA added.
For the last 20 years, the birds have been fed and watered by Gordon Knowles, also known as the chicken man, who noticed their numbers had dwindled.