The pets all stayed just doors apart in Eden Crescent, Glenrothes.
Now the Scottish SPCA has launched an appeal for information for what it called a “incredibly distressing” situation.
The charity said one cat started displaying symptoms on Monday, with a further cat passing away in similar circumstances a month ago and another still currently missing.
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Inspector Robyn Gray, said: “This has been an incredibly distressing, sad time for all of the owners involved and our thoughts are with them.
“One of the cats, Salem, was just 11 months old. Another cat, Toodles, was 10 and lived just a few doors away.
“Mini, who lived at another address, was two years old. Her owners lost one of their other cats in nearly identical circumstances just a month ago.
“They own a third cat who is currently missing from home and they are understandably now even more concerned about what might have happened.”She said all three cats had the same symptoms of vomiting, loss of coordination and balance.”
She added: “ Their vets have confirmed that the most likely cause is anti-freeze poisoning.
“Ethylene glycol is highly poisonous to cats and causes a very slow, painful death.
“As all three cats lived in close proximity to each other, we are concerned that we’re potentially dealing with a deliberate act and we are urging the public to report anything suspicious.
“However, it could be that these cats are accessing a garage or area where anti-freeze is not stored safely so we are appealing for local residents to please check anywhere that they think might be a risk.
“It is essential that everyone stores anti-freeze out of reach of cats and other animals and checks routinely to make sure there are no leaks.
“There are strict guidelines for use and anyone who uses this substance to cause deliberate harm is committing an offence.
“We’d also ask local cat owners to be vigilant and to seek urgent veterinary treatment if their cat appears unwell.
Call the SSPCA’s confidential animal helpline: 03000 999 999.