Gallbladder absorption increases during early cholesterol gallstone formation and is influenced by the intraluminal presence of lithogenic bile. The effect of lithogenic bile on gallbladder mucosal blood flow is unknown. The current study tested the hypothesis that the presence of lithogenic gallbladder and hepatic bile enhances gallbladder mucosal blood flow in cholesterol-fed (0.4%) prairie dogs, as determined by hydrogen gas clearance. Gallbladder mucosal blood flow in control animals was 35.57 +/- 3.9 mL.min-1.100 g-1. In contrast, basal gallbladder mucosal blood flow in cholesterol-fed animals was significantly (P less than 0.01) increased to 64.94 +/- 8.7 mL.min-1.100 g-1. In crossover studies, the addition of lithogenic gallbladder bile to control animals (n = 6) resulted in a significant (P less than 0.025) 26% increase in gallbladder mucosal blood flow, whereas the addition of nonlithogenic gallbladder bile into gallbladders of cholesterol-fed prairie dogs resulted in a significant (P less than 0.025) 58% decrease in gallbladder mucosal blood flow. Similarly, hepatic bile crossover studies showed that the addition of lithogenic hepatic bile to control gallbladders significantly increased (P less than 0.025) gallbladder blood flow by 30%, whereas instillation of nonlithogenic hepatic bile in gallbladders of cholesterol-fed animals significantly (P less than 0.025) decreased gallbladder mucosal blood flow by 29%. These results suggest that alterations in gallbladder mucosal blood flow, influenced by the presence and absence of lithogenic bile, may play a role in cholesterol gallstone formation.