Sphincterotomy has previously been shown to decrease gallbladder volume and gallbladder bile stasis while inhibiting gallstone formation in the prairie dog. We tested the possibility that atropine administration might reverse the effects of sphincterotomy on gallbladder mechanical function and gallbladder bile stasis and thereby reverse the inhibition of gallstone formation seen after sphincterotomy. Sixteen prairie dogs underwent sphincterotomy and were placed on a high cholesterol diet known to consistently induce gallstone formation. After 6 wk, in addition to the lithogenic diet, 8 animals were administered atropine. A control group of 8 animals was continued on the lithogenic diet. At death, animals that had undergone sphincterotomy and received atropine had a larger mean gallbladder volume (2.7 +/- 0.4 ml) than sphincterotomized control animals (0.66 +/- 0.2 ml, p less than 0.005), more total lipid stored in the gallbladder (176.1 +/- 33.9 mumol, sphincterotomy and atropine vs. 42.8 +/- 17.3 mumol, sphincterotomy, p less than 0.005), and a larger fraction of administered radiolabeled bile salt stored in the gallbladder (51.5 /- 7.3%, sphincterotomy and atropine vs. 24.5 +/- 10.7 sphincterotomy, p less than 0.05). The ratio of the fraction of [3H]cholic acid in the gallbladder (given 1 day before death) to the fraction of [14C]cholic acid in the gallbladder (given 4 days earlier) was lower for animals that underwent sphincterotomy and received atropine (0.63 +/- 0.04) than for sphincterotomized control animals (1.0 +/- 0.15, p less than 0.05), indicating a return of gallbladder bile stasis in the atropine-treated group. Confirming our previous work, only 1 of 8 animals that underwent sphincterotomy had gallstones. In contrast, 8 of 8 atropine-treated animals formed stones (p less than 0.005). Gallbladder bile of both groups were equally supersaturated with cholesterol. These findings provide direct support for the role of gallbladder bile stasis in the pathogenesis of cholesterol gallstone formation in this model.