Although the mechanism of carcinogenesis by hepatitis C virus (HCV) is not clearly known, core and NS3P protein have been shown to form tumors in specific cell lines. In this study, on the basis of the fact that the core and NS4B proteins of Kunjin virus translocate into the nucleus, we were prompted to investigate whether the HCV nonstructural protein NS4B has any function in tumor formation. First, we examined the location of the NS4B protein of HCV in transfected cells and then its oncogenic activity by transfection of NIH3T3 cells with the NS4B gene in the presence or absence of the Ha-ras gene. The NS4B protein was present only in the cytoplasm, particularly in the perinuclear region, different from the case of the Kunjun virus, The cells expressing HCV NS4B cooperatively with the Ha-ras gene showed loss of contact inhibition, morphological alterations, and anchorage-independent growth, These biological activities were confirmed by the transcription activation of the reporter gene from the AP1 promoter, by the NS4B protein in association with Ha-ras. Our results demonstrated that HCV NS4B protein in association with the Ha-ras gene played an important role in the malignant transformation of cells by HCV.