A Cryptosporidium parvum homogenate (CPH), prepared from partially excysted oocysts, was examined for proteolytic activity capable of hydrolyzing azocasein. Protease activity, measured at pH 7.0, was not detected in fresh oocysts but increased progressively with incubation at 37 C. Activity peaked after 60 min incubation but progressively decreased with extended incubation intervals. Cryptosporidial protease activity was significantly inhibited (P < 0.01) by the serine protease inhibitors phenylmethylsulfonyl fluoride (PMSF), diisopropyl fluorophosphate (DIFP), aprotinin, alpha-1-antitrypsin (AAT), and the cysteine protease inhibitor L-trans-epoxysuccinyl-leucylamido-(4-guanidino)-butane (E-64). No single inhibitor completely blocked CPH-associated protease activity; however, the combination of PMSF and E-64 inhibited > 95% of the azocasein hydrolysis measured in untreated control samples. The same group of inhibitors was further evaluated for their ability to inhibit excystation of C. parvum oocysts. PMSF, DIFP, aprotinin, and AAT significantly inhibited (P < 0.05) oocyst excystation at 15-, 30-, and 60-min incubation intervals; E-64 had no significant inhibitory effect on excystation. The results of this study demonstrate proteolytic activity during peak periods of excystation. Further, cryptosporidial protease activity was sensitive to both serine and cysteine protease inhibitors, but only serine protease inhibitors significantly inhibited oocyst excystation. These findings provide preliminary evidence of cryptosporidial proteases of both serine and cysteine protease classes and suggest that serine protease(s) are functionally associated with excystation.