Intravenous administration of 1 U cholecystokinin-pancreozymin (CCK-PZ) to rats caused the release of enteropeptidase, alkaline phosphatase (AP), and sucrase to the intestinal lumen in the absence of a concomitant increase in luminal DNA. Thus, the hormone elicited hydrolase secretion was not due to cell desquamation. Pentagastrin also stimulated hydrolase release. Following CCK-PZ administration enteropeptidase was released preferentially over sucrase and AP and showed a linear correlation with total protein output. The specific enteropeptidase activity was higher in the perfusate following secretion than in the mocosa. Enteropeptidase was found mainly in soluble form in both mucosa and perfusate; addition of bile following enteropeptidase release further increased its activity. In contrast, sucrase and AP were found mainly in insoluble form in both mucosa and perfusate and their specific activities were higher in the mucosa. The presence of bile rendered both sucrase and AP more soluble in the perfusate. The data indicate that enteropeptidase is released by a specific secretory process and that its subcellular site of origin is different from that of sucrase and AP. By eliciting the coordinated release of trypsinogen, enteropeptidase and bile, CCK-PZ plays a central role in the initiation of protein digestion.