The Scottish SPCA is appealing for information and urging cat owners to be vigilant following the alleged poisonings in Newtongrange, Midlothian.
Reports have been received of four possible cat poisonings in the past month, concentrated in and around the town's Fifth Street.
A five-year-old black cat called Lucky died on Monday after showing symptoms of poisoning.
Her owner, who lives on nearby Main Street but did not want to be named, said: "Lucky was very sleepy on Sunday night, and was sleeping under the covers, which she doesn't normally do.
"At 7am on Monday she was desperate to get outside, but by 11am one of the neighbours had called the SSPCA as she looked like she had a cold.
"The vets at Dalkeith Veterinary Surgery said that they thought it might have been anti-freeze poisoning.
"She was having seizures every 15 minutes, and her liver and kidneys failed, which led to her dying at 4pm."
A post-mortem examination is being carried out to establish the exact cause of death.
Scottish SPCA inspector Emma Phillips said: "We are concerned by the number of incidents which have been reported to us over a short space of time and in a very small location.
"This suggests there is a source of poison lying out in the area which cats have ready access to.
The vet who examined Lucky said the animal showed "advanced, untreatable symptoms" associated with poisoning.
The animal charity is urging local cat owners to be vigilant while the matter is investigated.
Animals which have consumed anti-freeze will display a number of symptoms including vomiting and lethargy, and in the latter stages head-shaking and coma, which ultimately results in renal failure and death.
Owners whose cats appear unwell should seek immediate veterinary treatment as early detection offers the best chance of survival.
Ms Phillips added: "At this early stage we do not know whether the poisonings are the result of a deliberate act or an accidental spillage so we are appealing to local residents to contact us if they have any information in relation to these concerning incidents."
Anyone with information should call the Scottish SPCA animal helpline on 03000 999 999. Calls will be treated in the strictest confidence.