Cats are essential in the epidemiology of Toxoplasma gondii because they are the only hosts that can excrete the environmentally resistant oocysts in nature. Samples of serum, feces, and tissues from feral cats from St Kitts, West Indies were examined for T. gondii infection. Antibodies to T. gondii were assayed by the modified agglutination test, and found in 71 of 96 (73·9%) of cats with titres of 1:10 in six, 1: 20 in six,1:40 in seven,1: 80 in three, 1: 160 in 10, 1:320 in 13, 1:640 in nine, and 1:1,280 or higher in 17. Tissues of 10 cats were bio-assayed in mice. Toxoplasma gondii was isolated from tissues of 7 cats; from hearts of 6, from tongue of 5, and brains of 3 cats. All 7 isolates were avirulent for mice. Toxoplasma gondii oocysts were not found in the feces of 51 cats. Genotyping of these 7 T. gondii isolates by 10 multi-locus PCR-RFLP markers, including SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and an apicoplast marker, Apico, revealed 4 genotypes, including clonal Type II, Type III and 2 unique genotypes. Five of the 7 cats had infection with 2 genotypes, indicating high frequency of mixed infection in the cat population on the St Kitts island.