The regulatory roles of intracellular taurine and glycine, transported and biosynthesized in hepatocytes, on the formation of conjugated bile acids were studied using freshly isolated hepatocytes (fresh hepatocytes) and hepatocytes in primary culture (cultured hepatocytes). Transported taurine significantly increased the rate of taurocholic acid formation both in fresh hepatocytes and cultured hepatocytes. Similarly, the addition of cysteine and hypotaurine, which were metabolically converted to taurine in hepatocytes, facilitated the formation of taurocholic acid in these cells. On the other hand, exogenous glycine into the incubation medium had no effect on the formation of glycocholic acid both in fresh and cultured hepatocytes. In contrast, the addition of serine and threonine, which are metabolically converted to glycine in hepatocytes, significantly increased the formation of glycocholic acid in fresh hepatocytes, although little effect of the additions of serine and threonine on the formation of glycocholic acid was noted in the case of cultured hepatocytes. The present results indicate that the formation of taurine-conjugated bile acids in hepatocytes is maintained by both transported and intracellularly formed taurine in hepatocytes, while that of glycine-conjugated bile acids is regulated by glycine formed within hepatocytes, but not by transported glycine.