Feeding and fasting influence biliary lipid composition. With total parenteral nutrition (TPN), it is possible to study the effects of a long-term "enteral fast" on biliary lipid composition without the metabolic ill-effects of nutrient deprivation. We compared the lipid and bile acid (BA) contents of hepatic and gallbladder biles in rabbits on completion of a 14-day regimen of TPN with those in rabbits returned to oral feeds for 6 weeks after a similar spell of TPN. Chow-fed rabbits served as controls. With TPN, plasma phospholipid and cholesterol levels were elevated. Basal bile flow and the secretion of bile acids and phospholipids were decreased in the TPN and post-TPN groups, while the cholesterol secretion rate was essentially unchanged. During TPN, the molar percent of cholesterol (relative to bile acids and phospholipid) in hepatic bile was increased. Biliary glycolithocholic acid (GLCA; as a percent of total conjugated BA) in hepatic bile increased from 1.7% (0.9% SEM) in the chow-fed to 8.5% (1.5% SEM) during TPN. In TPN and post-TPN groups, the gallbladder was enlarged to more than twice normal (chow-fed) size, and contained a dark, mucoid bile (biliary sludge). In this bile, (a) there was a 2.5-fold increase in bile acid concentration; and (b) the molar percent of cholesterol decreased while that of bile acids increased. TPN produced a state of functional cholestasis, which extended into the post-TPN period. Gallbladder distension was the common denominator of the hepatobiliary dysfunction in the TPN and post-TPN rabbits. With sequestration of bile acids in the gallbladder during and after TPN, the circulating bile acid pool was constricted, and the enterohepatic circulation impaired. As cholesterol secretion was low at all times, cholesterol supersaturation did not occur. The molar percent of cholesterol in gallbladder bile decreased, while that of bile acids increased; this suggests absorption of cholesterol by gallbladder mucosa. The increase in biliary GLCA probably resulted from bacterial biotransformation of glycochenodeoxycholic acid to lithocholic acid and its increased absorption from the cecum during TPN.