The synthesis of bile acids by primary hamster hepatocytes in culture has been studied. Measurable rates of bile acid synthesis were obtained from cells prepared from livers of animals fed 2% w/w cholestyramine to induce the synthesis of bile acids through the rate-limiting enzyme cholesterol 7 alpha-hydroxylase. The effects of various sources of substrate for bile acid synthesis in these cultured cells were examined over a period of 24 h and the results compared with published or parallel studies in primary rat hepatocytes or in the human hepatoma cell line, HepG2. In all the cells, bile acid synthesis was stimulated by the addition of 7 alpha-hydroxycholesterol, indicating the rate-limiting role of the cholesterol 7 alpha-hydroxylase. Bile acid synthesis in the hamster hepatocytes was also stimulated by a variety of sources of cholesterol as substrate, mevalonic acid (increasing the production of newly-synthesised cholesterol in the cell), and as an exogenous source, hamster LDL. Similarly, if cholesterol was diverted from intracellular esterification using the ACAT inhibitor Dup128, a further increase in bile acid synthesis could be demonstrated. These results show that hepatocytes obtained from cholestyramine-treated hamsters are deficient in substrate cholesterol for bile acid synthesis. A similar conclusion can be drawn from the published work with rat hepatocytes and is further supported by experiments on the regulation of cholesterol 7 alpha-hydroxylase activity at the mRNA and the protein level, although some in vivo studies in animals and studies in man have led authors to suggest that cholesterol 7 alpha-hydroxylase is saturated with substrate.