Studies were designed to examine skin test responses to leishmanial antigens in American visceral leishmaniasis (AVL) in Brazil. We found that after recovery from AVL, patients had positive delayed hypersensitivity reactions to Leishmania. Different amounts of a soluble extract obtained from Leishmania donovani chagasi promastigotes were compared with whole L. d. chagasi promastigotes in persons with past AVL. The most effective soluble preparations tested contained 25 and 50 µg leishmanial protein. These produced positive responses in 95%–100% of the individuals with past AVL. The 25 µg protein dose was used in further studies. This preparation produced no positive responses in either normal controls, tuberculosis patients, or schistosomiasis patients, and <5% positive responses in persons with Chagas' disease. The same amount of soluble extract prepared from L. mexicana amazonensis produced 82% positive skin test responses in persons with past AVL. When persons living in an area endemic for AVL were skin tested with the 25 µg preparation of L. d. chagasi extract, 34.1% yielded positive tests with a low number of positive responses in young children and 48% positive in adults. Only 3.1% of the population studied had a history of AVL. We have found that positive delayed hypersensitivity response to a soluble Leishmania extract is a sensitive and specific indicator of previous infection with AVL.