The purpose of this work was to determine which parameters trigger expression of proteins that are potentially important for the differentiation of Leishmania mexicana from the promastigote to the amastigote stage. To this effect, a protein-free axenic incubation system was used that supported the differentiation of L. mexicana promastigotes into amastigotes at 33 degreesC and at acidic pH. The predominant modification detected in SDS-PAGE patterns of extracted soluble proteins was the appearance in parasites cultured for 4 days of a strong 28-kDa protein band that displayed the same position and intensity as seen in amastigotes extracted from a mouse lesion. These molecules exhibited in gelatin gels the typical lytic pattern of cysteine proteinases (CPs) and were shown to belong to the CPb family, as further demonstrated by N-terminal amino acid sequencing. The expression of these enzymes was quantified by their lytic activity on the fluorogenic Z-F-R-AMC CP substrate. When the parasites were incubated at 33 degreesC for 3 days at various initial pHs, CPb started to be induced when the pH dropped below 5. When comparing cultures maintained at 26 or 33 degreesC for 3 days, it was seen that a rise in extracellular proton concentration (to pH 4.2-4.6) resulted in production of CPb at both temperatures (around 20-fold over the concentration measured in promastigotes cultured at 26 degreesC, pH >6). These results demonstrate that extracellular proton concentration is a key regulator of cysteine proteinase CPb synthesis and that an increase in temperature is neither necessary nor sufficient for the expression of this enzyme.