Major etiologic factors associated with human hepatocellular carcinomas (HCCs) include infection with hepatitis C (HCV) and hepatitis B virus (HBV), excess alcohol intake and aflatoxin B1 exposure. While the G→T p53 mutation at codon 249 has been identified as a genetic hallmark of HCC caused by aflatoxin B1, the genetic profile associated with other etiologic factors appears to be less distinctive. In our study, we screened HCCs resulting from HCV infection (51 cases), HBV infection (26 cases) or excess alcohol intake (23 cases) for alterations in genes involved in the RB1 pathway (p16INK4a, p15INK4b, RB1, CDK4 and cyclin D1), the p53 pathway (p53, p14ARF and MDM2) and the Wnt pathway (β-catenin, APC). Alterations of the RB1 pathway, mainly p16INK4a methylation, loss of RB1 expression and cyclin D1 amplification, were most common (69–100% of cases). There was a significant correlation between loss of RB1 expression and RB1 methylation. All 24 HCCs with RB1 promoter methylation lacked RB1 expression, while none of the 67 cases with RB1 expression exhibited RB1 methylation (p < 0.0001), suggesting that promoter methylation is a major mechanism of loss of RB1 expression in HCCs. Alterations of the p53 pathway consisted mostly of p53 mutations or p14ARF promoter methylation (20–48%). Mutations of the p53 gene were found at a similar frequency (13–15%) in all etiologic groups, without any consistent base change or hot spot. Mutations of β-catenin were found in 13–31% of cases, while no APC mutations were detected in any of the HCCs analyzed. With the exception of only 3 of 39 cases (8%), cyclin D1 amplification and β-catenin mutations were mutually exclusive, supporting the view that cyclin D1 is a target of the Wnt signaling pathway. Overall, the RB1, p53 and Wnt pathways were commonly affected in HCCs of different etiology, probably reflecting common pathogenetic mechanisms, i.e., chronic liver injury and cirrhosis, but tumors associated with alcoholism had more frequent alterations in the RB1 and p53 pathways than those caused by HCV infection. © 2003 Wiley-Liss, Inc.