Screening by enzyme electrophoresis of isolates of New World Leishmania from different geographic areas revealed a number of stocks with enzyme profiles different from those produced by reference strains of described subspecies of L. mexicana, L. braziliensis, and L. donovani. Analysis by six enzymes (aspartate aminotransferase; alanine aminotransferase; malate dehydrogenase; glucose-6-phosphate dehydrogenase; phosphoglucomutase; and glucose-phosphate isomerase) showed that these stocks have identical enzyme profiles and form a distinct zymodeme grouping. These observations were confirmed using the technique of schizodeme analysis and by comparing the k-DNA fingerprints produced by the restriction enzymes MspI, BspRI and AluI. The stocks were further analyzed by monoclonal antibodies and did not react with any of a large panel of L. mexicana, L. braziliensis, and L. donovani species- and/or subspecies-specific monoclonal antibodies using either an indirect radioimmune binding assay or immunofluorescence. These stocks did, however, react with a panel of monoclonal antibodies specific for L. major (formerly L. tropica major). Furthermore, the stocks could not be differentiated from L. major reference strains by enzyme electrophoresis nor could they be distinguished qualitatively from L. major based on their reactivity patterns using 10 Old World cutaneous species- and subspecies-specific monoclonal antibodies. Kinetoplast DNA restriction enzyme profiles, however, were different between these stocks and L. major reference strains. The implications of these results are discussed including the existence of other L. major-like stocks currently misidentified or uncharacterized.