The balance between hepatic glucose uptake and production is perturbed in both major forms of diabetes. It has been suggested that pharmacologic or genetic methods for enhancing glucokinase (GK) enzymatic activity in liver might be a means of increasing glucose disposal and lowering blood glucose in diabetic patients. To better evaluate this possibility, we used a recombinant adenovirus containing the cDNA encoding GK (AdCMV-GKL) to achieve overexpression of the enzyme at different levels in liver of normal rats. In a first set of experiments, in rats fasted for 18 h, AdCMV-GKL infusion caused a 211% increase in hepatic GK activity relative to animals infused with a control virus (AdCMV-betaGAL). AdCMV-GKL-treated fasted rats exhibited no significant changes in circulating glucose, free fatty acids (FFAs), lactate, beta-hydroxybutyrate, or insulin levels relative to controls, whereas triglyceride (TG) levels were slightly increased (53%). In a second set of studies, in rats fed ad libitum, GK was overexpressed in liver by 3- and 6.4-fold. Animals with the lower degree of GK overexpression exhibited no significant changes in circulating glucose, FFAs, insulin, TG, or lactate levels relative to controls that received a virus encoding a catalytically inactive mutant GK (AdCMV-GK203), but did show a modest increase in lactate (58%) relative to AdCMV-betaGAL-infused controls. In contrast, the higher level of GK overexpression caused a 38% decrease in blood glucose levels and a 67% decrease in circulating insulin levels relative to AdCMV-GK203-infused animals. The decline in glucose levels was accompanied by a 190% increase in circulating TG and a 310% increase in circulating FFAs; total plasma cholesterol was unaffected. Finally, fasted animals treated with AdCMV-GKL had 5.4 times as much liver glycogen as AdCMV-betaGAL-treated controls; no significant increases in liver glycogen were observed at either level of GK overexpression in ad libitum-fed rats relative to fed controls. In sum, levels of hepatic GK overexpression associated with a decline in blood glucose are accompanied by equally dramatic increases in FFAs and TG, raising concerns about manipulation of liver GK activity as a viable strategy for treatment of diabetes.