1. The synthesis of conjugated chenodeoxycholic acid and tauro-beta-muricholic acid in isolated rat hepatocytes was measured by radioimmunoassay. Production of tauro-beta-muricholic acid was linear over 4 h of incubation at 37 degrees C. The net synthesis of conjugated chenodeoxycholic acid was very much lower than that of tauro-beta-muricholic acid. 2. When hepatocytes were prepared from rats in which the enterohepatic circulation had been broken, either by feeding the bile salt sequestrant, cholestyramine or by total biliary drainage for 48 h, synthesis of tauro-beta-muricholic acid was increased compared to that in cells from control rats. Conjugated chenodeoxycholic acid accumulation during incubation of the hepatocytes was increased by cholestyramine feeding but not by total biliary drainage. These results suggest that there is a metabolic difference between the two methods of breaking the enterohepatic circulation with regard to chenodeoxycholic acid synthesis. 3. Hepatocytes prepared from rats given 1% cholesterol in the diet for at least 2 weeks synthesised significantly more tauro-beta-muricholic acid than those from control rats. The total amount of conjugated cholic, chenodeoxycholic and tauro-beta-muricholic acids synthesised by cells from cholesterol fed animals, however was not significantly different from that produced by hepatocytes from normal rats. 4. Exogenous taurochenodeoxycholic acid was metabolised to tauromuricholic acid by isolated hepatocytes. Production of tauro-beta-muricholic acid reached a maximum at a concentration of 20 microM taurochenodeoxycholic acid. The total metabolism of taurochenodeoxycholic acid, however, increased linearly up to the highest concentration measured, 50 microM. 5. The biliary content of tauro-beta-muricholic acid during total biliary drainage fell rapidly in the first 10 h and thereafter continued to decline, reaching a minimum after about 24 h. No significant rise was observed during the remainder of the experimental period. 6. It is concluded that a large proportion of the conjugated chenodeoxycholic acid synthesised by isolated hepatocytes in vitro is metabolised to tauro-beta-muricholic acid, and therefore it is necessary to take this into account when using this system to study bile salt synthesis.