To determine the role of cholecystokinin and the cholinergic system in intestinal stimulation of gallbladder contraction, we studied the effects of atropine on plasma cholecystokinin and gallbladder contraction in six healthy volunteers (four men and two women aged 20 to 27 yr). Effects were noted after intraduodenal fat instillation and after dosage with exogenous cholecystokinin inducing plasma cholecystokinin concentrations similar to those after intraduodenal fat instillation. At regular intervals before and after administration of each stimulus, plasma cholecystokinin concentrations and gallbladder volumes were measured by radioimmunoassay and real-time ultrasonography, respectively. Intraduodenal infusion of 250 ml 20% Intralipid induced a peak plasma cholecystokinin increment of 10.2 ± 1.6 pmol/L compared with 10.7 ± 0.7 pmol/L during infusion of 1 Ivy dog unit per kilogram per hour of cholecystokinin. The increases in plasma cholecystokinin after fat and exogenous cholecystokinin administration were accompanied by similar decreases in gallbladder volume. Integrated gallbladder contraction after fat instillation was 3,939% ± 288%. min compared with 3,301% ± 359%. min during cholecystokinin infusion (NS). Atropine (0.015 mg/kg as bolus followed by 0.005 mg/kg/hr) did not change plasma cholecystokinin concentrations but induced similar inhibition of gallbladder contraction to 2,296% ± 511%. min (p < 0.05) after intraduodenal fat instillation and to 1,756% ± 456%. min (p < 0.05) during cholecystokinin infusion. We conclude that cholecystokinin is of major importance in intestinal stimulation of gallbladder contraction. Atropine inhibits the gallbladder response to intraduodenal fat. This inhibition is not due to a reduction in cholecystokinin secretion but to a diminished gallbladder response to cholecystokinin.