The objective of this study was to compare, in rat small intestinal and colonic enterocytes, subcellular distributions of activities degrading the large peptides, neurotensin, acetylneurotensin (8-13), GRF(1-29)NH2 (human growth hormone releasing factor fragment), (desNH2Tyr1,D-Ala2,Ala15)-GRF(1-29)NH2, insulin, and insulin B-chain. Proteolytic activities degrading individual peptides in the 10,000-g pellet, rich in intracellular organelles, 27,000-g pellet, rich in brush-border membrane, 100,000-g pellet, and 100,000-g supernatant, rich in cytosol, were determined and compared for both the small intestine and colon. In colonic fractions, the cytosol had highest activity (g protein)-1 degrading three out of four peptides tested, while in small intestinal fractions, the 27,000-g pellet had the highest activity (g protein)-1, degrading four out of five peptides tested. In both small intestine and colon, the cytosol had a higher percentage of total proteolytic activity degrading each of the above polypeptides and the highest insulin-degrading activity (g protein)-1. The results suggest that at pH 7.5, proteolytic activities (g protein)-1 in the fraction of subcellular organelles are much lower than those in cytosol and that cytosolic proteolytic activities degrading polypeptides and analogues are significant.