CYP2B, CYP4A, and CYP2E1 mRNA levels are elevated in response to pathophysiological conditions, such as diabetes, high-fat diet, and fasting, in which lipids and ketone bodies are increased. In order to avoid confounding hormonal effects, we utilized primary rat hepatocytes to examine whether ketone bodies or fatty acids altered CYP2B, CYP4A, or CYP2E1 expression. Ketone bodies increased CYP2B mRNA and protein levels, but failed to alter CYP4A or CYP2E1 expression. Straight-chain saturated fatty acids, C8 to C16, increased levels of CYP2B and CYP4A mRNA, but not CYP2E1 mRNA. Treatment with octanoylcarnitine, a mitochondrial beta-oxidation inhibitor, in combination with hexadecanoate increased CYP2B and CYP4A expression approximately 1.4-fold over that observed with hexadecanoate alone, suggesting that mitochondrial conversion of fatty acids to ketone bodies was not required for enhanced CYP2B expression and that mitochondrial beta-oxidation decreased intracellular fatty acid levels and thereby lowered CYP2B expression. Undecynoic acid or aminobenzotriazole treatment increased CYP2B mRNA levels, consistent with these compounds inhibiting the initial CYP4A-catalyzed step in the conversion of monocarboxylic to dicarboxylic acids and thereby decreasing peroxisomal beta-oxidation and increasing intracellular fatty acid levels. Addition of glycerol, which suppresses fatty acid synthesis by inhibiting conversion of lactate to pyruvate, decreased basal expression of CYP2B and CYP4A but did not alter CYP2E1 expression. Pyruvate, but not lactate, completely prevented the glycerol-mediated decrease in CYP2B expression. These results provide evidence that intracellular levels of fatty acids and ketone bodies regulate the expression of CYP2B but not CYP2E1.