Isoelectrofocusing in agarose was used to characterize Leishmania spp. isolated from a kala-azar patient, a lizard, genet cat and elephant shrew. Isoenzyme profiles of 12 enzymes and general protein patterns were examined. Both methods were found to be useful for distinguishing between the isolates. The wild animal Leishmania spp. were shown to be indistinguishable from a reference strain of L. major, but the isolate from man was distinctly different from L. donovani sensu stricto. The unique finding that the lizard isolate was similar to L. major and its significance are discussed in some detail. The use of the isoelectrofocusing technique for separation of enzymes and general proteins for characterization purposes is emphasized.