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Five hundred thirty stocks of Leishmania isolated from human and domestic and wild reservoir hosts, representing a wide geographic distribution of endemic foci of American cutaneous (ACL) and visceral leishmaniases (AVL) were characterized and identified at species and/or subspecies levels based on their reactivity to a cross-panel of specific monoclonal antibodies using a radioimmune binding assay. This study confirms and extends our preliminary results on the high specificity of some of these monoclonals for the L. braziliensis, L. mexicana, and L. donovani complexes. This study also demonstrates the relative stability of these molecular markers and the general usefulness of the method for parasite identification. Two hundred ninety-two of 420 isolates of ACL were classified as members of the L. braziliensis complex. Two hundred twenty-seven were L. b. braziliensis; these showed the widest geographical distribution (Brazil: Amazonas, Bahia, Ceara, Espirito Santo, Goias, Minas Gerais, Para, Paraiba, Rio de Janeiro, and Sao Paulo; Honduras: Santa Barbara and Yoko; Peru: Ancash, Piura, and Ucayali; and Venezuela: Cojedes, Distrito Federal, Lara, Portuguesa, Vale Hondo, Yaracuy, and Zulia). Forty-one stocks were identified as L. b. guyanensis (from North Brazil: Amazonas, Amapa, Para, and Rondonia). Twenty-one stocks were identified as L. b. panamensis (from Costa Rica: Alajuela, Guanacasten, Limon, Puntarenas, and San Jose; and Honduras: El Paraiso, and Olancho). Out of 128 isolates classified as members of the L. mexicana complex, 74 were differentiated as L. m. amazonensis (from Bolivia; Brazil: Amazonas, Bahia, Ceara, Goias, Maranhao, Mato Grosso do Norte, and Para; Peru: Pasco Forest and Van Humboldt; and Venezuela: Carabobo, Guarico, and Merida). Forty-four stocks were identified as L. m. venezuelensis (from Venezuela: Lara). Six stocks were L. m. mexicana (from Belize; and Mexico: Campeche [corrected] and Quintana Roo, Yucatan). One hundred ten isolates from AVL were identified as L. donovani chagasi (from Brazil: Bahia, Ceara, Maranhao, Minas Gerais, Mato Grosso do Sul, Piaui, Rio de Janeiro, and Sergipe; and Honduras: Valle). The implications of these results with respect to both the clinical and epidemiological data (including the detection of seven unusual characterized stocks) are discussed.