Police say a dead buzzard found in woods near Ballingry had been poisoned.
It had been thought that the bird had died of natural causes but toxicology tests carried out by a Scottish Government laboratory this week confirmed the presence of a banned pesticide.
The area where the bird was found in April is just a short distance from the Loch Leven nature reserve.
Detective Inspector Colin Robson, who is leading the inquiry, said: “The bird was found on land in an area commonly used by dog walkers. From the toxicology results, I believe it is probable that the bird has fed on an animal carcass deliberately laced with this poison.
“Such an act is both illegal and highly irresponsible in an area regularly used by members of the public, and the placing out of poison baits like this is indiscriminate in its victims.
“This illegal chemical is highly toxic, and the ingestion of even minute quantities by a wild animal or a pet is likely to have fatal consequences.
“Although relatively remote, I would urge anyone who frequents this area to contact the police if they saw anyone or anything suspicious around this time or have knowledge of this or similar incidents.
“Since the discovery there have been no reported linked incidents locally and the area where the bird was found has been searched and there has no trace of the poison or other carcasses.
“Police Scotland are committed to tackling wildlife crime and this matter is being robustly investigated in close collaboration with partner agencies.”
Ian Thomson, Head of Investigations at RSPB Scotland said: “The recent incident on the Black Isle, where 22 birds of prey were killed, showed very clearly the horrendous impact that the illegal use of poisons can have on wildlife.
“It is of great concern that someone has placed a bait laced with this illegal chemical out in the countryside in an area well-used by the public and close to our own nature reserve at Loch Leven.
“I urge anyone who has information about this incident to contact the police as soon as possible.”
Brent Meakin, Forestry Commission Scotland’s district manager for the Lowlands, said: “It is appalling that individuals are carrying out this illegal and barbaric practice.
“The persecution of raptors must stop. Any poisoning of these birds is one too many, no matter the species. We will continue to work with the Police and other agencies to stamp out this activity.
“The commission would also like to ask the public for their help as they too can be our eyes and ears and report any suspicious activity.”