Functional inactivation of tumor suppressor genes during tumor progression has been shown to occur by either coding region mutation or promoter region methylation. Because of the functional equivalence of these two mechanisms, loss of tumor suppressor function generally occurs by one or the other mechanism, but rarely by both. Aberrant de novo methylation in most tumor suppressor promoter regions is found within CpG islands that occur near the transcription start site. The p53 promoter region is unique in that it does not contain a CpG island and therefore it is possible that methylation at critical CpG sites may be more important in gene silencing than total CpG methylation density. Other than site-specific aflatoxin B(1)-induced mutations, p53 coding region mutations are not frequently observed in most human primary hepatocellular carcinomas. In the present study, paired samples of human primary liver carcinoma and uninvolved tissue obtained from the same individual were evaluated for site-specific p53 promoter methylation status by methylation sensitive single nucleotide primer extension (Ms-SNuPE) and also for coding region mutations using polymerase chain reaction (PCR)- single strand conformation polymorphism (SSCP). The methylation pattern in the uninvolved tissue was variable at specific CpG sites, whereas the same sites had become highly methylated in tumor tissue from the same individual. Associated with de novo methylation, the level of p53 mRNA was significantly reduced in the tumor DNA relative to the uninvolved tissue DNA. None of the samples exhibited coding region mutations. Given that p53 mutations are rare in primary human liver tumors, these data suggest that transcriptional repression by p53 promoter methylation may contribute to tumor progression.