We used methylation-sensitive restriction fingerprinting (MSRF) to identify novel CpG (5'-CG-3' palindrome; p, phosphate group)–rich sequences that are methylated differentially between the hepatocellular carcinoma (HCC) genomes and adjacent nontumorous liver tissues. A 199-base-pair sequence methylated in HCC tumor tissue was isolated and showed high homology to the 5'-CpG island of the zinc fingers and homeoboxes protein 2 (ZHX2) gene. By using bisulfite sequencing, we confirmed that hypermethylation of the 5'-CpG island of ZHX2 occurred in some HCC and HepG2 cell lines but not in 6 normal liver tissue samples. By using methylation-specific polymerase chain reaction, we detected methylation of the 5'-CpG island of ZHX2 in 46.9% of the HCCs. Reverse transcription–polymerase chain reaction demonstrated that ZHX2 messenger RNA (mRNA) was expressed in all 6 normal liver tissue samples but in only 13.3% of the methylated HCCs. Treatment of HepG2 with 5-aza-deoxycytidine could demethylate the promoter and increase ZHX2 mRNA expression. These results suggest that hypermethylation-mediated silencing of ZHX2 is an epigenetic event involved in HCC.