This study was designed to determine which morphologic form and species of Leishmania is most suitable for detection of antibody in sera from American visceral leishmaniasis (AVL) patients by the indirect fluorescent antibody test (IFAT). Promastigotes and amastigotes of Leishmania mexicana or Leishmania donovani chagasi were used as sources of antigen. A total of 70 sera, including 30 from AVL patients, 30 from healthy subjects and 10 from Chagas' disease patients, were used in the study. Titers of antibody up to a dilution of 1:64 were found with all four antigens. At a titer of 1:128, the sensitivity of the IFAT using L. d. chagasi promastigotes as a source of antigen was 100% and the specificity at a titer of 1:32 was 98%. Although the sensitivity of the amastigote forms was close to 100% at a similar titer, the specificity at a titer of 32 using the L. d. chagasi amastigotes was 91% and using L. mexicana amastigotes was only 80%. The L. d. chagasi promastigote antigen was also the one that showed less cross reaction with sera from Chagas' disease patients. Since cross reactivity between Trypanosoma cruzi and Leishmania species is well known in serological tests, and minimizing of such cross reactivity is of critical importance for diagnosis, we suggest that L. d. chagasi promastigotes should be the antigen of choice for diagnosis of visceral leishmaniasis by IFAT in areas also endemic for trypanosomiasis.