A highly specific and sensitive competitive serodiagnostic assay for visceral leishmaniasis (VL) was developed using species specific Leishmania donovani monoclonal antibodies. This assay, either RIA or ELISA, is based on the specific inhibition of monoclonal antibody binding to a crude parasite homogenate by serum from patients with VL. 15 monoclonal antibodies were examined. The binding of 13 antibodies was significantly inhibited by VL serum and unaffected by normal serum. 3 species-specific monoclonal antibodies, D-2, D-13 and D-14, which recognize different parasite antigens, were chosen for use in the competitive serodiagnostic assay. In 90% of the positive cases, regardless of geographic origin, VL sera inhibited monoclonal antibody binding to the parasite antigen by more than 30%. No false positive was obtained with sera from Chagas disease, lepromatous leprosy, schistosomiasis, malaria, systemic lupus erythematosus, cutaneous or mucocutaneous leishmaniasis, even at serum dilutions (1:100) which cross-react strongly with Leishmania antigen in direct binding assays. Inhibition by negative control sera from areas endemic for VL and from non-endemic areas was negligible. The assay takes less than 24 h, requires minimum amounts of sera or antigen, and is easily standardized allowing interlaboratory comparison of test data. The competitive serodiagnostic assay will be especially useful in areas where Chagas disease is coendemic and the rapid diagnosis of VL by direct binding serodiagnostic assays presents a problem.