We report herein a specific, sensitive, and rapid bioassay for measuring plasma cholecystokinin in rats. Plasma was first passed through octadecylsilylsilica cartridges and the extracts were then tested for their content of cholecystokinin, based on their ability to stimulate amylase release from isolated rat pancreatic acini. Plasma levels of cholecystokinin-octapeptide as low as 0.18 pM were detectable. Gastrin, in contrast, reacted only weakly in this system. Cholecystokinin bioactivity was inhibited by the antagonist dibutyryl cyclic guanosine monophosphate and was eliminated by immunoadsorption with an antibody directed against the carboxyl terminus of cholecystokinin. Plasma cholecystokinin levels in fasting rats as cholecystokinin-octapeptide equivalents were 0.31 +/- 0.05 pM (mean +/- SE) and rose to 6.2 +/- 1.8 pM after feeding. Plasma cholecystokinin levels also increased 30-fold after intragastric instillation of soybean trypsin inhibitor and 15-fold after ethanol instillation. After column chromatography of plasma, two different forms of cholecystokinin were identifiable; one eluted with the octapeptide of cholecystokinin whereas the other most abundant form was intermediate in size between cholecystokinin-33 and cholecystokinin-octapeptide.