We have established previously a mouse strain containing a mutant β-catenin allele of which exon 3 was sandwiched by loxP sequences [Catnb^lox(ex3)]. In this mouse strain, a Wnt-activating β-catenin mutation alone is insufficient for hepatocarcinogenesis, but additional mutations or epigenetic changes may be required. Here we report that hepatocellular carcinoma develops at the 100% incidence in mice with simultaneous mutations in the β-catenin and H-ras genes that are introduced by adenovirus-mediated Cre expression. Although H-ras mutation alone rapidly causes large cell dysplasia in the hepatocytes, these cells show no autonomous growth within 1 week after infection of the Cre-adenovirus. However, simultaneous induction of an additional mutation in the β-catenin gene causes a clonal expansion of such dysplastic cells, followed by nodular formation and development of hepatocellular carcinoma. These results indicate that β-catenin mutations play a critical role in hepatocarcinogenesis in cooperation with another oncogene and that these mice provide a convenient model to investigate early steps of hepatocarcinogenesis.