Diabetes has been reported to increase the expression of cytochrome P450 (CYP) 2E1 messenger RNA (mRNA) and protein several-fold, and enhanced expression has been associated with elevated ketone bodies. Primary cultured rat hepatocytes were used to explore ketone body and insulin regulation of CYP2E1 expression. Hydroxybutyrate and acetoacetate (AC), alone or in combination, either failed to affect or decreased CYP2E1 mRNA levels by up to 90% relative to untreated hepatocytes. Insulin produced a concentration-dependent decrease in CYP2E1 mRNA levels, and insulin receptor immunoprecipitation showed a correspondence between receptor phosphorylation and the decrease in CYP2E1 mRNA levels at physiologic levels of insulin. Phosphatase inhibitors decreased CYP2E1 mRNA levels by greater than 95%. The phosphatidylinositol 3-kinase (PI3-kinase) inhibitors wortmannin or LY294002 and rapamycin, an inhibitor of p70 S6 kinase phosphorylation, ameliorated the insulin-mediated decrease in CYP2E1 mRNA levels. Geldanamycin, which inhibits Src kinase, also abrogated the insulin-mediated decrease in CYP2E1 mRNA levels. In contrast, the protein kinase C (PKC) inhibitor bisindolylmaleimide, the mitogen-activated protein kinase kinase (MEK) inhibitor PD98059, and the p38 mitogen-activated protein (MAP) kinase inhibitor SB202190 did not affect the insulin-mediated decrease in CYP2E1. CYP2E1 mRNA half-life decreased from approximately 48 hours in the absence of insulin to approximately 15 hours at 10 nmol/L insulin, and this decrease was prevented by wortmannin. The half-life of CYP2B mRNA was increased by insulin, whereas that of CYP3A was unaffected. Analysis of CYP2E1 gene transcription using heterogeneous nuclear RNA (hnRNA) showed that insulin suppressed CYP2E1 transcription. In conclusion, these data show involvement of transcriptional and posttranscriptional mechanisms in the insulin-mediated regulation of CYP2E1 and implicate PI3-kinase, p70 S6 kinase, and Src kinase in mediating these effects.