Dietary fat has been found to alter the incidence of cholesterol gallstones in hamsters: butterfat intensifies while safflower oil reduces lithiasis. WE not report how dietary fat affects bile flow and biliary lipid secretion in this model. Male hamsters were fed one of three experimental diets: a control diet (containing 0.3% cholesterol); control diet + 4.0% butterfat; or control diet + 4.0% safflower oil. After three weeks, bile samples were collected via an external biliary fistula. The endogenous bile acid pool was depleted for 120 min followed by increasing rates of taurocholate infusion for 160 min. Basal secretion of biliary lipids was measured during the bile acid depletion period. Basal bile flow and bile acid output were not significantly different in the three groups. Dietary butterfat increased basal cholesterol output compared to the control diet (0.037 vs. 0.025 mumol/min.kg, respectively); safflower oil did not change cholesterol output (0.027 mumol/min.kg). Hamsters fed butterfat or safflower oil secreted more phospholipid (0.171 and 0.178 mumol/min.kg, respectively) than controls (0.131 mumol/min.kg). The cholesterol/phospholipid output ratio of the butterfat group was higher than the safflower oil group (0.220 vs. 0.153, respectively). Effects of dietary fat on several relationships between file flow and biliary lipid secretion were analyzed by linear regression using the data for the entire bile collection period (bile acid depletion and taurocholate infusion). Butterfat and safflower oil did not change either bile acid dependent or bile acid independent bile flow. Hamsters fed butterfat had a higher linkage coefficient (slope) of cholesterol vs. bile acid output than the safflower oil group (0.023 vs. 0.009, respectively). The linkage coefficient of phospholipid vs. bile acid output of the butterfat group was higher than the controls (0.278 vs. 0.185, respectively). In summary, butterfat induced a high cholesterol and phospholipid secretion with a high cholesterol/phospholipid output ratio; safflower oil induced a high phospholipid secretion with a low cholesterol/phospholipid output ratio. Butterfat and safflower oil have different effects on biliary lipid secretion. These differences in biliary lipid secretion may explain, in part, how butterfat and safflower oil differ in affecting gallstone formation in hamsters.