Leishmanial organisms isolated from 24 patients with Andean cutaneous leishmaniais (uta) and from 7 with sylvatic leishmaniasis in both cutaneous and mucosal forms were characterized on the basis of their isoenzyme profiles for 13 enzymes using both cellulose acetate (CA) and thin-layer starch gel (TLS) electrophoretic techniques. Malate dehydrogenase (MDH) after electrophoresis on CA or TLS and mannose phosphate isomerase (MPI) on TLS were the only enzymes of 13 examined which discriminated between the organisms from patients with uta (L. (V.) peruviana) and those with sylvatic leishmaniasis (L. (V.) braziliensis). Mannose phosphate isomerase gave more clear-cut and reproducible discrimination than did MDH on either TLS or CA, and it is suggested that MPI is a reliable enzyme marker that can be used in routine TLS electrophoresis to distinguish between L. (V.) peruviana and L. (V). braziliensis. © 1990, Royal Society of Tropical Medicine and Hygiene. All rights reserved.