American cutaneous leishmaniasis (ACL) is an endemic disease in the north-west of Paraná state, Brazil, where it occurs in humans and dogs. Few studies have been undertaken on the occurrence of the disease in other domestic animals and horses. In this study we investigated the infection of horses by Leishmania in ACL-endemic rural areas. Direct agglutination test (DAT) and polymerase chain reaction (PCR) were employed. Fifty-five horses from rural areas in the municipalities of Doutor Camargo, Ourizona, São Jorge do Ivaí, Ivatuba and Santa Fé (Agua do O) were analysed. DAT-detected antibody titres varied from 10 to 640, and 42 (76.3%) animals presented titres > or =20. PCR analyses led to the detection of Leishmania (Viannia) DNA in the blood samples of three (7.1%) of 42 animals. The presence of anti-L. (V.) braziliensis antibodies and Leishmania (Viannia) DNA in horses from endemic areas suggests their participation in the ACL transmission cycle.