Antibodies to Toxoplasma gondii were determined in 167 mammals in three zoos in Mexico City, Mexico, using the modified agglutination test (MAT). Overall, antibodies to T. gondii were found in 89 (53.3%) of the 167 animals tested. Antibodies were found in 35 of 43 wild Felidae: 2 of 2 bobcats (Lynx rufus); 4 of 4 cougars (Puma concolor); 10 of 13 jaguars (Panthera onca); 5 of 5 leopards (Panthera pardus); 7 of 7 lions (Panthera leo); 2 of 3 tigers (Panthera tigris); 2 of 3 ocelots (Leopardus pardalis); 2 of 2 Sumatran tigers (Panthera tigris sumatrae); 1of 2 Jaguarundi (Herpailurus jagouaroundi); but not in 0 of 2 oncillas (Leopardus tigrinus). Such high seroprevalence in wild felids is of public health significance because of the potential of oocyst shedding. Four of 6 New World primates (2 of 2 Geoffroy's spider monkeys [Ateles geoffroyi], 1 of 3 Patas monkeys [Erythrocebus patas], and 1 of 1 white-headed capuchin [Cebus capucinus]) had high MAT titers of 3,200, suggesting recently acquired infection; these animals are highly susceptible to clinical toxoplasmosis. However, none of these animals were ill. Seropositivity to T. gondii was found for the first time in a number of species.