Epimastigotes of different stocks of Trypanosoma cruzi contain levels of proteinase activity on azocasein; amastigotes and trypmastigotes contain 10-fold lower levels of this proteolytic activity, which seems, therefore, to be developmentally regulated. The proteinase could be detected as a broad band, centered at about 60 kDa, which in some resolved into two close bands, in (a) SDS-polyacrylamide gels containing fibrinogen, and (b) Western blots probed with polyclonal rabbit antiserum prepared against purified cystein proteinase. No proteinase activity was observed at molecular weights lower than 55 kDa. The results show that the enzyme preciously purified is teh major cysteine proteinase present in epimastigotes of all stocks of T. cruzi tested.