Two electrophoretically distinct trypsins and chymotrypsins and an elastolytic enzyme were isolated from rat pancreatic juice. Rabbit antisera against these enzymes were produced, and with an immunochemical technique the trypsins, chymotrypsins, and elastase were studied in the intestinal contents of conventional and germfree rats. In both types of rat the anionic trypsin and chymotrypsin were the most abundant and found in higher concentrations in the distal than in the proximal small intestine. The cecal and fecal concentrations of anionic trypsin were markedly higher in the germfree rat when compared to the conventional rat. Cymotrypsin was undetectable in the large intestine of either the conventional or germfree rat when this technique was used. Immunoreactive elastase was found in greater amounts in the distal small intestine, and high concentrations were demonstrated in the cecal contents and feces of the germfree rat. In contrast, no immunoreactive elastase was detected in the large intestine of the conventional rat. Gel filtration indicated that the immunoreactive anionic trypsin and elastase found in fecal extracts were of about the same molecular size as the native enzymes. The findings suggest that the intestinal microflora is instrumental in the inactivation and degradation of pancreatic trypsin and elastase but not chymotrypsin.