Clostridium difficile pathogenicity is related to in vivo production of toxins, and it is of great interest to detect toxins produced in biological samples. Several reports have shown that proteases in stools interfere with immunological methods for quantitation of toxin A. The purpose of this work was to estimate the relationship between the proteases and the C. difficile toxins produced in a gnotobiotic mouse model of pseudomembranous cecitis. Cecal proteolytic activities hydrolyzed toxin A, and immunoglobulin G bound to the microtiter plate used in immunoassays. This interference could be blocked by the addition of trypsin inhibitor to the samples. The ability of soluble toxin A to bind to bound antibodies in an enzyme-linked immunosorbent assay was not affected by the proteases, but the biological activity was reduced 100-fold. The cytotoxicity of toxin B was not modified by proteolytic activity treatment. Mice inoculated with a low toxin A-producing strain of C. difficile did not died, and no modulation of proteolytic activities occurred. After inoculation with the lethal VPI strain of C. difficile, toxins A and B reached maximum levels in the ceca at 12 h postinfection. At this time, the proteolytic activities did not decrease from the levels seen at zero time. Mice died within 2 days. At this time (about 32 postinfection), proteolytic activities were sharply decreased in the lower parts of the digestive tracts. The findings that serum inhibited the proteases and that there was a 100-fold increase in serum-derived mouse immunoglobulins in the lumen as the C. difficile infection progressed suggest that the decrease in protease activity in the lower digestive tract may be related to the exudation of serum from the inflammation process.