An extracellular serine peptidase was purified 460-fold from Trypanosoma cruzi epimastigotes culture supernatant with (NH(4))(2)SO(4) precipitation followed by affinity chromatography aprotinin-agarose and continuous elution electrophoresis, yielding a total recovery of 65%. The molecular mass of the active enzyme estimated by reducing and non-reducing SDS-PAGE was about 75kDa. The optimal pH and temperature of this glycosylated peptidase were 8.0 and 37 degrees C using alpha-N-rho-tosyl-L-arginine-methyl ester (L-TAME) as substrate. The enzyme did not hydrolyze polypeptide substrates but was active against short peptide substrates containing arginine at the P1 site, in both ester and amide bonds. The peptidase was inhibited by TPCK and TCLK but not by other protease inhibitors suggesting that the enzyme belongs to the serine peptidase class. Interestingly, the enzyme seems to demonstrate some metal dependence since its activity was reduced by 1,10-phenanthroline, calcium and zinc ions. Rabbit anti-T. cruzi extracellular serine peptidase antiserum was used to show that the enzyme was restricted to intracellular structures, including the flagellar pocket, plasma membrane and cytoplasmic vesicles resembling reservosomes. These results suggest that the serine oligopeptidase is secreted into the extracellular environment through the flagellar pocket and the intracellular location could suggest its participation in certain proteolysis events in reservosomes. These findings show that this peptidase is a novel T. cruzi serine oligopeptidase, which differs not only from other peptidases described in the same parasite but also in other species of Trypanosoma.