Antibodies to Neospora caninum were assayed in sera of 222 female water buffaloes from Ribeira Valley of São Paulo State, Brazil, using an indirect fluorescent antibody test (IFAT) and Neospora agglutination test (NAT). IFAT antibodies were found in 64% of buffaloes with titers of 1:25 (42 buffaloes), 1:50 (53 buffaloes), 1:100 (31 buffaloes), 1:200 (10 buffaloes), 1:400 (3 buffaloes), or > or =1:800 (3 buffaloes). NAT antibodies were found in 53% of buffaloes; in titers of 1:40 in 52 buffaloes, 1:80 in 27 buffaloes, 1:160 in 21 buffaloes, and > or =1:320 in 17 buffaloes. Results indicate a high prevalence of N. caninum exposure in water buffaloes in Brazil and warrant an investigation of the role of N. caninum as an abortifacient in water buffaloes.