The effect of dietary fat on conjugated cholic, chenodeoxycholic and tauro-beta-muricholic acid synthesis was studied using hepatocytes isolated from rats given a low-fat diet, or a low-fat diet mixed with 10% olive oil or 10% corn oil. The rats were totally biliary drained for 48 h prior to preparation of the cells in order to raise bile salt synthesis to a level which was measurable by radioimmunoassay. Synthesis of both conjugated cholic and chenodeoxycholic acid was raised in hepatocytes from rats given a fat supplement (either corn oil or olive oil) in the diet as compared to that in cells from low-fat-fed animals. Tauro-beta-muricholic acid synthesis, however, was unaffected by corn oil feeding. Production of conjugated cholic acid was increased to a greater extent when rats were given olive oil as opposed to corn oil, but these differences were not statistically significant. The conjugated cholic, chenodeoxycholic, and tauro-beta-muricholic acid and cholesterol content of bile collected at 2-h intervals during the biliary drainage of the same groups of rats was also determined. The pool size of both conjugated cholic and chenodeoxycholic acid in the enterohepatic circulation was found to be significantly decreased in rats given olive oil as compared to those given corn oil or the low-fat diet only. The pool size of tauro-beta-muricholic acid was also decreased in the olive oil-fed rats compared to the other two groups, but this difference was not statistically significant. After the pool had been drained out, animals which had received fat in the diet secreted more conjugated cholic and chenodeoxycholic acid into the bile than rats which had received the low-fat diet only. This effect was more marked when the fat given was olive oil rather than corn oil. Secretion of tauro-beta-muricholic acid into bile at this stage of biliary drainage was not changed by dietary fat supplements. Biliary cholesterol excretion was also increased in rats on diets containing 10% fat, with olive oil again having a greater effect than corn oil. The results show that supplementing the diet with fat leads to increased synthesis of conjugated cholic and chenodeoxycholic acids and biliary cholesterol secretion in the rat. The relatively more saturated fat, olive oil (85% oleate), gave a consistently larger increase than the more unsaturated, corn oil (50% linoleate), but the type of fat appeared less important than the presence of fat in the diet.