Sera from diverse clinical forms of American cutaneous leishmaniasis show remarkably different patterns of reactivity to Leishmania mexicana and L. braziliensis after absorption with these two species of Leishmania. The enhanced species specificity of absorbed sera was confirmed by reactions with well characterized reference strains. The use of pairs of enriched polyclonal sera from mucocutaneous and diffuse cutaneous leishmaniasis permits the preliminary classification of isolates in the L. mexicana or L. braziliensis complexes by a simple, inexpensive, and quantitative immunoassay.