To elucidate a role of glucokinase in hepatic glucose metabolism, we overexpressed hexokinase I (HKI), liver type glucokinase (LGK), or β cell type glucokinase (βGK) in primary rat hepatocytes using a recombinant adenovirus vector system. Overexpression of HKI and LGK induced a 34- and 25-fold increase, respectively, in glucose phosphorylation activity measured in cell homogenates. While HKI overexpression induced only a 1.3-fold increase in glucose oxidation, LGK overexpression increased glucose oxidation by 2.9-fold. Overexpression of βGK had essentially the same effect as LGK. The results indicate that glucokinase does indeed regulate the rate of hepatic glucose oxidation and that the liver-specific sequence of this enzyme is not essential for this function.