We studied gallbladder bile flow before, during, and after cholesterol gallstone formation in the prairie dog using infusion cholescintigraphy with ^99mTc-diethyl iminodiacetic acid. In 18 fasting animals partitioning of bile between gallbladder and intestine was determined every 15 min for 140 min, and gallbladder response to cholecystokinin (5 U/kg · h) was calculated from the gallbladder ejection fraction. Ten prairie dogs were then placed on a 0.4% cholesterol diet and 8 on a regular diet, and the studies were repeated 1, 2, and 6 wk later. The proportion of hepatic bile that entered the gallbladder relative to the intestine varied from one 15-min period to the next, and averaged 28.2% ± 5.1% at 140 min. Partial spontaneous gallbladder emptying (ejection fraction 11.5% ± 5.6%) was intermittently observed. Neither the number nor the ejection fraction of spontaneous gallbladder contractions changed during gallstone formation. By contrast, the percent of gallbladder emptying in response to cholecystokinin decreased from 72.1% ± 5% to 25.9% ± 9.3% (p < 0.025) in the first week and was 14.3% ± 5.5% at 6 wk (p < 0.01 from prediet values, not significant from first week). Gallbladder filling decreased from 28.2% ± 5.1% to 6.7% ± 3% (p < 0.01), but this change was only observed after 6 wk, when gallstones had formed. This study shows that (a) bile flow into the gallbladder during fasting is not constant; (b) the gallbladder contracts intermittently; (c) gallbladder emptying in response to exogenous cholecystokinin is altered very early during gallstone formation; and (d) gallbladder filling remains unaffected until later stages, when gallstones have formed.