Isolated hepatocytes synthesize fatty acids and cholesterol from lactate and acetate with lactate being the more effective substrate. Biotin deficiency decreased fatty acid synthesis from both substrates but stimulated cholesterogenesis. Exposure of intact hepatocytes to oxalate inhibited fatty acid and cholesterol synthesis from lactate, this effect was enhanced in biotin-deficient chicks. A similar effect was not observed when acetate was the substrate. Synthesis of fatty acids from lactate and acetate was stimulated by glucose, biotin deficiency increased this response. Cholesterogenesis was reduced in control but not biotin-deficient chicks.