Because of prior difficulties in measuring plasma cholecystokinin (CCK) levels, it has not been established which components of food stimulate CCK secretion in rats. In the present study, we used a sensitive and specific bioassay for measuring plasma CCK and determined the effects of proteins, protein hydrolysates, amino acids, fats, starch, and glucose on CCK secretion in this species. Intact proteins were the only stimulants of CCK release. Solutions of 18% casein and 0.2% soybean trypsin inhibitor caused prompt increases in plasma CCK levels from 0.5 +/- 0.2 to 7.9 +/- 1.9 and 8.0 +/- 2.0 pM, respectively, within 5 min of orogastric administration. The proteins lactalbumin and bovine serum albumin caused smaller elevations in circulating CCK. In contrast, hydrolysates of casein and lactalbumin and the amino acids L-phenylalanine and L-tryptophan did not stimulate CCK release. In addition, plasma CCK levels did not increase with the feeding of fat, starch, or glucose. The ability of proteins to stimulate CCK secretion paralleled their ability to inhibit trypsin activity in vitro. Furthermore, the plasma CCK response to casein was completely abolished by the simultaneous administration of trypsin. These studies indicate that proteins are the major food stimulants of CCK release in the rat and that the effects of proteins are related to inhibition of intraluminal protease activity.