Seroprevalence of Toxoplasma gondii was determined in 865 captive neotropical felids from 20 states from Brazil, sampled from September 1995 to April 1997. Sera were tested by the modified agglutination test (MAT) using formalin-fixed whole tachyzoites and mercaptoethanol. Antibodies (MAT> or =1:20) to T. gondii were found in 472 of 865 (54.6%) cats: in 45 of 99 (45.9%) jaguarundis (Herpailurus yagouaroundi), in 97 of 168 (57.7%) ocelots (Leopardus pardalis), in 68 of 131 (51.9%) oncillas (L. tigrinus), in 35 of 63 (55.5%) margays (L. wiedii), in 1 of 8 (12.5%) Pampas-cat (Oncifelis colocolo), in 9 of 12 (75.0%) Geoffroys-cat (O. geoffroyi), in 134 of 212 (63.2%) jaguars (Panthera onca), and in 83 of 172 (48.2%) pumas (Puma concolor). Antibody titers were: 1:20 in 27 felids, 1:25 in 142 felids, 1:40 in 6 felids, 1:50 in 292 felids, and > or =1:500 in 5 felids. The high seroprevalence of T. gondii antibodies found in the present study suggested a widespread exposure of neotropical cats to T. gondii in zoos in Brazil. The results warrant an investigation on the mode of exposure and oocyst shedding by neotropical cats.