Cats are important in the epidemiology of Toxoplasma gondii because they are the only hosts that excrete environmentally resistant oocysts in feces. In the present study, hearts, serum, and feces from 36 feral cats from Addis Ababa area, Ethiopia, were examined for T. gondii infection. Antibodies to T. gondii were determined with the modified agglutination test (MAT, cutoff 1:25); 33 cats were seropositive. Hearts of all 36 cats were homogenized, digested in pepsin, and bioassayed in mice. Feces were examined for T. gondii oocysts by bioassay in mice. Viable T. gondii was isolated from heart of 26 by bioassay in mice and from 25 seropositive and 1 seronegative cats. Toxoplasma gondii was isolated from feces (oocysts) by bioassay in mice. In total, viable T. gondii was isolated from 27 of the 36 cats, and these isolates were designated TgCatEt1 to TgCatEt27. The high prevalence of T. gondii oocysts in feces of 8 (19.4%) of 36 cats is of high epidemiologic significance. This is the first report of isolation of viable T. gondii from any host in Ethiopia.