We tested the possibility that ablation of the sphincter of Oddi might alter gallbladder mechanical function and thereby affect gallstone formation. Twenty-three prairie dogs underwent either sham duodenotomy or sphincterotomy. After a recovery period, 11 sphincterotomy and 12 sham-operated prairie dogs started a high-cholesterol diet known to consistently induce gallstone formation. At death, after 6 wk on the lithogenic diet, sphincterotomy animals had a smaller mean gallbladder volume (0.3 ± 0.1 ml) than sham-operated controls (1.2 ± 0.2 ml, p < 0.005), less total lipid (bile salt + phospholipid + cholesterol) stored in the gallbladder (68.6 ± 21.3 μmoles, sphincterotomy vs. 181.3 ± 46.2 μmoles, controls, p < 0.025), and a smaller fraction of administered radiolabeled bile salt stored in the gallbladder (26.3 ± 13.5%, sphincterotomy versus 62.5 ± 8.5%, controls, p < 0.025). The ratio of fraction [3H]cholic acid in the gallbladder (given 1 day before death) per fraction [14C]cholic acid in the gallbladder (given 4 days earlier) was higher for sphincterotomy animals (0.81 ± 0.06) than control animals (0.66 ± 0.07, p = 0.062), suggesting a reduction in gallbladder bile stasis by sphincterotomy. As expected, 10 of 12 sham-operated animals had gallstones. In contrast, only 1 of 11 sphincterotomy animals formed stones (p < 0.005). Cholesterol crystals were present in the gallbladder bile of all sham-operated animals and in 8 of 11 sphincterotomy animals. Gallbladder bile of both sphincterotomy and sham-operated animals were equally supersaturated with cholesterol (lithogenic index 2.02 ± 0.27 sphincterotomy vs. 2.39 ± 0.44 control). We conclude that sphincterotomy inhibits gallstone formation in this model despite continued supersaturation of bile. The mechanism may be alleviation of gallbladder bile stasis (known to precede gallstone formation in this model) with a reduction of gallbladder storage time below that required for crystal growth and agglomeration.