In order to clarify the significance of DNA methylation in both earlier and later stages of hepatocarcinogenesis, the DNA methylation state on chromosome 16, on which loss of heterozygosity (LOH) has frequently been detected in human hepatocellular carcinomas (HCCs), was examined. DNA from primary HCCs and tissues showing chronic hepatitis and liver cirrhosis, which are considered to be precancerous conditions, was analyzed by digestion with methylation-sensitive and non-sensitive restriction enzymes. DNA hypermethylation at the D16S32, tyrosine aminotransferase (TAT) and D16S7 loci and hypomethylation at the D16S4 locus were detected in 18%, 58%, 20% and 48% of examined HCCs, respectively. Aberrant DNA methylation occurred more frequently in advanced HCCs than in early HCCs. Moreover, DNA hypermethylation at the D16S32, TAT and D16S7 loci was frequently observed in chronic hepatitis and liver cirrhosis. The incidence of DNA hypermethylation was higher than that of LOH (42% at the TAT locus). These data suggest that DNA hypermethylation might predispose the locus to allelic loss. Aberrant DNA methylation is a significant change which may participate in the early developmental stages of HCCs.