Cyclin D1, one of the G(1) cyclins, is frequently overexpressed in several types of carcinomas and is thought to play an important role in tumorigenesis and tumor progression including hepatocellular carcinoma. We constructed a retrovirus vector-carrying rat cyclin D1 cDNA in the reverse orientation, resulting in expression of antisense (AS) cyclin D1 mRNA. For efficient transduction of this recombinant retrovirus, two-step gene transfer was performed. The rat hepatoma cell line (dRLh84) was infected with this recombinant retrovirus after preinfection with adenovirus expressing the retrovirus receptor. In the rat hepatoma cells, AS cyclin D1 mRNA was expressed, inducing a decrease in the expression of endogenous cyclin D1 mRNA and an inhibition of cell growth. Moreover, two-step gene transfer of AS cyclin D1 into s.c. hepatoma xenografts resulted in inhibition of tumor growth and prolonged animal survival. In the virus-infected tumor xenografts, expression of cyclin D1 was immunohistochemically inhibited, and apoptosis of hepatoma cells was detected. These findings suggest that transduction of AS cyclin D1 is useful as an adjunct to standard treatments for hepatocellular carcinoma.