Amastigotes of Leishmania species belonging to the Leishmania mexicana complex exhibit large lysosomes, called megasomes, which are rich in cysteine proteinases. Various aspects of the host-parasite interaction, the differentiation process as well as intracellular survival, have been attributed to these proteinases. The in vitro differentiation from promastigote to amastigote forms of Leishmania amazonensis was evaluated using the analysis of the expression of cysteine proteinase (Lpcys2) by Northern blot, Western blot and enzymatic activity. As promastigotes transformed to amastigotes, there was an increase in Lpcys2 RNA transcription, as well as an increase in Lpcys2 production and activity. Moreover, the processing rate of the cysteine proteinase precursor forms was also increased in amastigote forms. These results are in agreement with our previous study in which megasome development was demonstrated by morphometric and immunochemical analysis.