Many characteristics of sterol metabolism in the hamster resemble those in the human more closely than those in the rat, although the latter has been widely used as an experimental model for human cholesterol metabolism [ 11. For example, the hamster carries more of its plasma cholesterol in the low density lipoprotein fraction, and manifests much lower rates of bile acid production and whole body and hepatic cholesterol synthesis than the rat [ 11. Furthermore, like the human and unlike the rat, the hamster is able to produce lithogenic bile in response to various dietary manipulations (21. The hamster, therefore, might be expected to be a better experimental model for human cholesterol metabolism than the rat. Recent experiments in our laboratory have used isolated hamster hepatocytes in monolayer culture to investigate bile acid synthesis [3]. The aim of this study is to further characterize cholesterol metabolism in cultured hamster hepatocytes by studying the pathways of cholesterol synthesis, esterification and the hydrolysis of cholesteryl esters in addition to bile acid production. Hamster hepatocytes were prepared from the livers of male Golden Syrian hamsters fed a commercial diet, and maintained in monolayer culture for up to 24h as described previously 131. After the initial 2h of culture when the cells had adhered to the dishes, the medium was removed and replaced with fresh, serum-free medium. This represents time zero in all experiments. De MIVO synthesis of cholesterol was investigated by incubating the hepatocyte monolayers with 12-3HI mevalonic acid lactone or [ 114CI acetate for 3h, starting at various times over the 24h culture period. Radioactivity incorporated into unesterified cholesterol was then determined after extraction of the cells and medium with chlorofodmethanol ( 2 lvlv) and separation of the lipids by thin layer chromatography. Incorporation from both radiolabels was detected at all time points tested. In both cases a statistically significant decrease of approximately 25% was observed after the first 3h of culture, but thereafter incorporation remained relatively constant for up to 24h. Addition of mevinolin (2pg/ml), an inhibitor of the rate limiting enzyme for cholesterol synthesis, 3 hydroxy 3 methylglutaryl Co-enzyme A reductase (HMG CoA reductase), to the culture medium totally inhibited the incorporation of radioactivity from 11-14CI acetate, but not from 12-3Hl mevalonic acid lactone, into unesterified cholesterol. Thus cholesterol synthesis in cultured hamster hepatocytes appears to be regulated by HMG Co A reductase in the expected manner. Cholesterol esterification was investigated at various times during the 24h culture period by measuring the incorporation of radioactivity from I I l W I oleate into cholesteryl esters. The hepatocyte monolayers were incubated with potassium I 1 I T I oleate for 3h as described previously for experiments with cultured rat liver cells 141. The hamster cells esterified cholesterol at all time points tested, and the rate of esterification remained constant over the 24h culture period (figure I). Hydrolysis of cholesteryl ester was determined by assaying the neutral cholesteryl ester hydrolase activity in the hepatocyte monolayers at different times up to 24h. The post-mitochondria1 supernatant prepared from the cells by differential centrifugation 141 was used, and the assay was carried out as described in 1-51. except that the substrate, cholesteryl [ I-IJCI oleate, was delivered in acetone. Cholesteryl ester hydrolysis was observed at all time points tested, but decreased significantly over 24h (Figure I) . Previous work in our laboratory has shown that glycocholic and glycochenodeoxycholic acids are the major bile acids produced by hamster hepatocytes in monolayer culture 131. The mass of these two bile acids associated with the hepatocyte monolayers (cells + medium) at various times up to 24h was measured using radioimmunoassays 16.7). No increase in mass during 24h culture “f