Utilization of stearate as compared to various saturated fatty acids for cholesterol and lipid synthesis and beta-oxidation was determined in primary culture of rat hepatocytes. At 0.5 mmol/L in the medium, stearate (18:0) adequately solubilized by albumin was less inhibitory to cholesterol synthesis from [2-14C] acetate than myristate (14:0) and palmitate (16:0) (68% vs. 91 and 88% inhibition, respectively). The rate of incorporation into cholesterol from [1-14C] stearate (3.0 +/- 0.6 nmol/mg protein/4 h) was 37-, 1.8-, and 7.8-fold of that from myristate, palmitate, and oleate, respectively. Conversely, the rate of [1-14C] stearate incorporation into total glycerolipids was 88-90% lower than that of labeled palmitate, myristate, and oleate. The rate of [1-14C] stearate incorporation into triacylglycerol (3.6 +/- 0.4 nmol/mg protein/4 h) was 6-8% of that from myristate, palmitate, oleate, and linoleate. The rate of stearate incorporation into phospholipids was the lowest among tested fatty acids, whereas the rate of mono- and diacylglycerol synthesis was the highest with stearate treatment. The rate of beta-oxidation as measured by CO2 and acid soluble metabolite production was also the lowest with [1-14C] stearate treatment at 22.7 nmol/mg protein/4 h, which was 35-40% of those from other [1-14C] labeled fatty acids. A greater proportion of stearate than other fatty acids taken up by the hepatocytes remained free and was not metabolized. Clearly, stearate as compared to shorter-chain saturated fatty acids was less efficiently oxidized and esterified to triacylglycerol in cultured rat hepatocytes.