Recent studies indicate that gallbladder absorption increases during the early stages of experimentally-induced cholesterol gallstone formation. The purpose of the present study was to ascertain whether pharmacologic inhibition of gallbladder ion transport and absorption reduces the incidence of experimentally-induced cholesterol gallstones. Prairie dogs were fed either a control chow or a 1.2% cholesterol-enriched chow for 15 days. One group of cholesterol-fed animals received saline via an orogastric tube; another group received amiloride, a drug known to inhibit in vitro ion transport in the prairie dog gallbladder. The incidence of gallstones in cholesterol-fed animals was reduced from 83% to 13% (p less than 0.025) when the animals were treated with amiloride; this occurred despite a cholesterol-saturation index comparable to that observed in gallstone animals. Additionally, although biliary calcium decreased in the gallbladder, hepatic bile did not in the amiloride-treated animals. These data provide further evidence that altered gallbladder absorption and increased biliary calcium are important factors in the pathogenesis of cholesterol gallstones.