Inactivations of DNA repair genes, O(6)-methylguanine-DNA methyltransferase (MGMT) and hMLH1, by promoter hypermethylation have been reported in several types of primary human neoplasia. This epigenetic inactivation mechanism remains elusive in hepatocellular carcinoma (HCC). To investigate the relation between the expression of MGMT and hMLH1 and the CpG methylation within their promoters in HCCs with or without hepatitis viral infection, we performed immunohistochemistry and urea/bisulphite sequencing on 46 HCCs, corresponding noncancerous tissues, and 20 normal liver tissues. MGMT- and hMLH1-negative HCCs were 60.9% (28 out of 46) and 21.8% (10 out of 46), respectively. HCCs lacking both proteins were 10.9% (five out of 46). The frequency and extent of CpG methylation in the MGMT promoter increased along with hepatitis viral infection and pathological progression. MGMT-negative tumours showed very frequent and widespread methylation in the promoter compared with MGMT-positive tumours. Half of the hMLH1-negative HCCs showed promoter hypermethylation. These data suggested that MGMT gene silencing in a subset of HCCs was likely caused by epigenetic alteration, such as promoter hypermethylation, and that the promoter hypermethylation silenced the hMLH1 gene in half of the hMLH1-negative tumours. A correlation between the promoter methylation status and viral infection, although it was weak, intimated that hepatitis viral infections could play a role in the CpG methylation of the MGMT promoter.