The canine visceral leishmaniasis (CVL) and american trypanosomiasis are important zoonoses in public health and dogs are the main domestic reservoir of the parasite for humans. The goal of this study was to estimate the prevalence of circulating antibodies anti-Trypanosoma cruzi and anti-Leishmania sp. in sera of dogs from the rural area of Botucatu, SP, Brazil. During the annual vaccination campaign against canine rabies in rural area, 689 blood samples were taken and processed by indirect immunofluorescent antibody test. The serological tests revealed the absence of antibodies anti-Leishmania spp., but anti-T. cruzi antibodies were detected in 3 (0.4%) dogs.