High rate of chronic hepatitis B virus (HBV) infection and p16 promoter methylation were found in the majority of hepatocellular carcinoma (HCC). To investigate the potential linkage between high rate of p16 methylation and HBV infection, p16 methylation was detected with methylation-specific polymerase chain reaction (PCR), and HBV markers were examined with real-time PCR and immunologic method. p16 methylation was detected in 5.5% of patients with hepatitis B, 9.1% of noncancerous liver, 36.6% of cirrhotic liver tissue, and 70.5% of cancerous tissue of HCC, primarily in cirrhotic (46.7%) and cancerous tissue (90.6%) with HBV infection. In noncancerous tissue, p16 methylation could only be detected in samples with HBV infection, although no significant difference, the frequency of p16 methylation in noncancerous tissue with HBV infection was higher than those without it. The results showed that, in cancerous, cirrhotic, or noncancerous tissues, the frequency of p16 methylation in samples with HBV infection was higher than those without it, suggesting possible association between HBV infection and p16 methylation. The result of HBV-DNA analysis showed that 96.1% (49/51) samples with p16 methylation also showed detectable HBV-DNA; it signifies that replication and/or integration of HBV may contribute to high rate of p16 methylation in hepatocarcinogenesis. Generally, these results indicate that persistent HBV infection may be associated with high rate of p16 methylation, and involved in development of HCC through this way.