Chronic diversion of pancreatic and biliary secretions away from the proximal small intestine results in pancreatic hypertrophy in adult rats. Serum levels of cholecystokinin (CCK) were measured in age-matched control and surgically diverted rats at various times after operation by a radioimmunoassay method that was specific for the sulfated form of CCK. The concentration of CCK was markedly increased in bypassed rats as compared with controls. The increases in circulating CCK in bypassed rats was substantiated by a bioassay method that measured physiologically active CCK. The degree of pancreatic hypertrophy and the increase in CCK levels both progressed with time up to 23 days after surgery. Linear regression analysis showed an apparent direct correlation between pancreatic weights and serum CCK levels (r = 0.99). Feeding bypassed rats with diets containing various pancreatic and biliary supplements did not abolish the hyperplastic response of their pancreata. However, feeding with diets supplemented with bile partially suppressed the increase in serum CCK levels, while a diet containing Cotazyme and bile completely suppressed this increase. The discrepancy between serum CCK levels and the degree of pancreatic hypertrophy in the supplemented bypassed rats was further demonstrated by the lack of correlation using linear regression analysis (r = 0.33). The observed pancreatic hypertrophy in the absence of high serum levels of CCK in the bypassed rats fed bile and Cotazyme supplements suggests that serum hypertrophic factors other than CCK may also be involved in the enteral feedback regulation of pancreatic growth.