This study was undertaken to observe the effects of hydrogen peroxide on Cryptosporidium parvum oocysts with respect to protease activity in comparison to known protease inhibitors. In assessing the possible mechanisms of action of hydrogen peroxide, treatment effectiveness was analyzed using 3 assays and the potential roles of proteases and cations were considered. Treatment of C. parvum oocysts with hydrogen peroxide inhibited protease activity up to 50% compared with untreated controls. Treatment of oocysts with chemicals that affect sulfhydryls, including N-ethylmaleimide and dithiolthreitol, inhibited protease activity by > 90%. Treatment of oocysts with these chemicals, along with the protease inhibitors, phenylmethylsulfonyl fluoride (PMSF), ethylenediamine-tetraacetic acid, and cystatin, inhibited protease activity as well as in vitro excystation and infection in a cell culture assay. Several mechanisms may result in the successful inhibition of infection and excystation by hydrogen peroxide treatment, including: oxidation of oocyst wall proteins or lipids, chelating of cations necessary for infection, or hydroxyl radical-induced DNA damage to sporozoites, or both.