Serum samples from 77 animals belonging to 38 species and 19 families in Rio de Janeiro Zoo, Brazil were tested for antibodies against serovars of Leptospira interrogans by microscopic agglutination test. Antibodies were detected in serum samples of 37.7% of all animals belonging to 10 families. Seropositivity was more common in the Carnivora Canidae (7/9), Procyonidae (5/9) and the Edentata Myrmecophagidae (5/9). Icterohaemorrhagiae was the most common serogroup. Seropositivity was less common in primates.