Cysteine proteinase activities were examined in lesion amastigotes as well as in stationary-phase promastigotes of Leishmania (L.) mexicana and Leishmania (L.) amazonensis isolates. Enzyme detection in gelatin gels revealed that amastigotes of three L. (L.) mexicana isolates (M379, IOC-0561, and IP) shared similar proteinases, including the multiple low-molecular-weight (25-35 kDa) cysteine proteinases. High cysteine proteinase activity was also observed in L. (L.) amazonensis amastigotes, but the banding profile was different in two of the isolates examined. Promastigotes displayed fewer low-molecular-weight proteinase bands, and these were much less intense as compared with those of lesion amastigotes. Independently of the Leishmania isolates and developmental stages examined, incubation of the parasites for 2 h with 0.2 microM radioiodinated N-benzyl-oxycarbonyl-tyrosyl-alanyl diazomethane (Z-Tyr[125I]-AlaCHN2) markedly and selectively labeled bands comigrating with the 28- and 31-kDa cysteine proteinases. Under reducing conditions, labeling was associated with four similar polypeptides (29-34 kDa), which were also detected when incubation with Z-Tyr[125I]-AlaCHN2 was carried out after cell lysis. Labeling was completely abolished if lysates were first incubated with 20 microM E-64 and then exposed to the 125I-tagged inhibitor, thus confirming the specificity of the compound toward cysteine proteinases.