To locate regulatory element(s) that mediate(s) the effect of insulin on hexokinase II (HKII) gene transcription, we have generated several transgenic mouse lines harboring 97, 254, 505, 819 or 4077 bp of the proximal promoter of the human HKII gene driving the expression of a luciferase reporter gene. Luciferase activities indicate that major promoter elements responsible for the basal activity and tissue-specificity of the human HKII gene expression are located within the 505-bp segment of the promoter. To induce the promoter constructs by endogenous insulin released from the pancreatic beta-cells, transgenic mice were given repeated intraperitoneal injections of D-glucose. Significant induction of luciferase activity was not observed in any of the transgenic mouse lines even though the endogenous HKII mRNA was induced 2.7-fold upon treatment. Thus, our results suggest a lack of insulin response in the 4.0-kb region of the proximal promoter of the human HKII gene in mice.