Metabolic characteristics of experimental hepatoma cells include elevated rates of glycolysis and lipid synthesis. However, pyruvate derived from glucose is not redily oxidized, and the source of acetyl CoA for lipid synthesis in As-39D cells has not been characterized. In this study ketone bodies were examined as a possible source of acetyl CoA in AS-30D hepatoma cells. The major findings were: 1. Acetoacetate was utilized by AS-30D cells, with 14C-lipid and 14CO2 as major products of [3-14C] acetoacetate. 2. Lipid synthesis from acetoacetate was dependent on the presence of glucose in the medium. 3. Acetoacetate supported rapid respiration by AS-30D mitochondria in the presence of 0.1 mM malate. 4. Succinyl CoA acetoacetyl CoA transferase activity in AS-30D mitochondria was approximately 40 fold greater than that found in rat liver mitochondria. 5. Addition of acetoacetate, but not beta-hydroxybutyrate decreased conversion of [1-14C] acetate to 14CO2, presumably by diluting the specific radioactivity of the acetyl CoA derived from the acetate tracer. 6. In the presence of glucose, approximately one fourth of acetoacetate utilized was converted to lipid. This result is consistent with elevated lipogenesis postulated by the truncated TCA cycle hypothesis.(ABSTRACT TRUNCATED AT 250 WORDS)