Oral vanadate administration has been demonstrated to normalize blood glucose levels in ob ob and db db mice and streptozotocin (STZ) diabetic rats. The exact mechanism of this vanadate effect is uncertain, since there are no consistent effects on the insulin receptor tyrosine kinase activity or phosphotyrosine phosphatase activity. We have therefore studied the postreceptor actions of vanadate, focusing our attention on the steady-state levels of mRNA of enzymes involved in carbohydrate metabolism. When compared with their lean (ob/+) controls, the livers of ob ob mice exhibited an approximately 90% reduction in the levels of phosphoenolpyruvate carboxykinase (PEPCK) mRNA and twofold to fivefold higher levels of the mRNAs for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), the "liver β-cell" glucose transporter (GLUT2), and the proto-oncogene c-myc. Administration of sodium vanadate (0.25 mg/mL) in the drinking water of ob ob mice over a 45-day period resulted in a near normalization of blood glucose and increased PEPCK mRNA levels more than ninefold. Starvation of the ob ob mice for 24 to 48 hours also increased PEPCK mRNA levels by fourfold to 15-fold. Vanadate treatment did not alter mRNA levels of any other proteins studied and had no effect on PEPCK mRNA in ob/+ mice. However, 1 to 100 μmol/L vanadate produced a concentration-dependent increase in PEPCK mRNA levels in an H35 hepatoma cell line, an effect opposite to the suppression of PEPCK mRNA produced by insulin. In summary, hyperglycemia in the ob ob mouse is characterized by decreased expression of PEPCK and increased expression of GAPDH mRNA. PEPCK mRNA levels increase during fasting, suggesting that there is some regulatory effect by the high levels of circulating insulin that appear to ovecome, at least in part, the severe insulin resistance. Vanadate treatment increases the low PEPCK mRNA level as it normalizes blood glucose. The hypoglycemic effect of vanadate cannot be explained by any action on PEPCK at the level of mRNA expression, and in fact occurs despite an effect to increase PEPCK mRNA. Interestingly, ob ob mice do exhibit high levels of expression of GLUT2 in the liver, which may facilitate glucose efflux and thus contribute to increase hepatic glucose output. However, GLUT2 is unaffected by vanadate treatment, despite normalization of blood glucose.