The aim of the present study was to explore the relationship between methylation status of the p16(INK4A) promoter and some HBV-related factors, and the role of these factors in p16(INK4A) hypermethylation and hepatocellular carcinoma (HCC) progression. Twenty-three cases of surgically resected HBV-associated HCC and 25 fine-needle aspiration biopsy cases of chronic hepatitis B (CHB) were studied. The methylation status of the p16(INK4A) promoter was determined by methylation-specific polymerase chain reaction (PCR). Two-step immunohistochemical staining showed the expression of viral antigens in situ. Tissue HBV-DNA levels were determined by fluorescence quantitative real-time PCR. PCR and the direct sequencing method were used for mutation analysis. In peritumoral tissues (P = 0.025) and CHB samples (P = 0.029), the expression of hepatitis B virus X protein (HBx) was higher in methylated groups of p16(INK4A) promoter than in unmethylated groups. Other HBV factors including hepatitis B surface antigen and hepatitis B core antigen, tissue HBV-DNA levels and HBV x gene mutations had no relation to the methylation status of p16(INK4A) promoter. The data indicate that p16(INK4A) promoter hypermethylation correlated closely with higher HBx expression in the precancerous lesions, suggesting that HBx may play an important role in the early stage of HBV-associated hepatocarcinogenesis via induction of hypermethylation of p16(INK4A) promoter.