In order to evaluate the tumor-initiating activity of kojic acid (KA) in mouse liver, an in vivo initiation assay in liver was performed using partially hepatectomized mice. Male ICR mice were fed on a basal diet (BD) containing 0 or 3% KA for 4 weeks, followed by distilled water (DW) containing 0 or 500 ppm phenobarbital (PB) for 13 weeks. Two weeks after the treatment with PB, two-thirds partial hepatectomy was preformed in all mice in order to enhance the regeneration and proliferating activities of the hepatocytes. In microscopic examinations, no proliferative lesion was observed in any of the groups. There were no differences in the number of γ-glutamyltransferase-positive cells, an expected marker for preneoplastic hepatocytes in mice, between the KA + DW and the KA + PB groups. In the immunohistochemical analyses of the proliferating activity of hepatocytes, significant increases in the labeling index of proliferating cell nuclear antigen (PCNA) were observed in the BD + PB and KA + PB groups as compared to the BD + DW group; however, no significant difference in the positivity of PCNA was observed between the BD + PB and the KA + PB groups. These results of the present study suggest the possibility that KA has no tumor-initiating activity in the liver of mice.