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1. Rat intestinal segments have been incubated in isotonic saline in vitro and release of peptide hydrolase enzymes into the incubation media examined over a 90 min study period. 2. Chemical assay data, as well as analysis of electrophoretic mobilities of release enzymes on starch gel, indicate that peptide hydrolase enzymes in the incubation media originate predominantly from the cytoplasm of the mucosal cells. 3. Peptide hydrolases were released rapidly from intact intestinal segments. Release occurred from the start of the in vitro incubations and was not affected by temperature and shaking. 4. It is concluded that compared to the in vivo situation, cytoplasmic peptide hydrolases are released from intestinal tissue very rapidly in vitro. Caution is therefore required when comparing results of in vivo and in vitro peptide absorption experiments.