Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. Little is known about its molecular pathogenesis and the relevance of DNA methylation for disease initiation and progression. Nevertheless, promoter methylation of some genes has been implicated as potential marker for HCC. Thirty-four HCC, 34 matching non-malignant, cirrhotic livers and 16 normal livers were analyzed for the methylation status of the genes p16INK4a, GSTP1, MGMT, DAP-K and APC by quantitative methylation-specific PCR. DNA promoter methylation frequencies in HCC and matching non-malignant cirrhotic liver, respectively, were as follows: p16INK4a (76% vs. 24%), GSTP1 (53% vs. 32%), MGMT (6 vs. 12%), DAP-K (68 vs. 100%) and APC (100 vs. 100%). GSTP1 and/or p16INK4a promoter methylation was observed in 88% of the HCC samples. In normal liver tissue, the p16INK4a, GSTP1 and MGMT promoter were not methylated. DAP-K was methylated in 31% and APC even in 100% of normal liver samples. Quantitative levels of methylated promoter DNA of all genes were significantly different in the various tissue types except for MGMT. Our results suggest that promoter methylation of tumor-associated genes is a common event in hepatocarcinogenesis. Significantly, higher levels and frequencies of promoter methylation in HCC were found for p16INK4a and GSTP1 compared to non-malignant cirrhotic liver. This indicates that these epigenetic events may serve as a good marker for HCC. These data also demonstrate the importance of the quantification of methylated promoter DNA within a given sample and the use of normal tissue as controls. Quantitative analyses of methylated GSTP1 and p16INK4a promoter may serve as a powerful molecular marker in detecting HCC in biopsies. © 2008 Wiley-Liss, Inc.