The release of enterokinase into human duodenal fluid was studied after intravenous injections of secretin and cholecystokinin-pancreozymin (CCK-PZ). In five control subjects there was a significant release of the enzyme after stimulation with either hormone. A similar release of enterokinase was observed after hormonal stimulation in three patients with total biliary obstruction and in four patients with pancreatic exocrine insufficiency. These results suggest that the hormone-mediated release of enterokinase is independent of bile salts and trypsin in man. This release of enterokinase into duodenal fluid may be physiologically important in protein digestion.