Previous studies have established that chronic dietary insufficiency of the lipotropic nutrients choline and methionine with or without chemical initiation is hepatocarcinogenic in the rat and certain mouse strains. In the present study, the folate/methyl-deficient model of multistage hepatocarcinogenesis was used to evaluate progressive in vivo changes in p16 promoter methylation in both preneoplastic and tumor tissues. Previous studies using this model have demonstrated stage-dependent alterations in genome-wide and p53 gene-specific methylation. In the present study, we used highly sensitive methylation specific PCR (MSP) to determine time of appearance of methylated sequences within p16 promoter. In addition, methylation-sensitive single nucleotide primer extension methodology was applied to determine methylation status of the remaining CpG sites within amplified methylated alleles. Using this approach, extensive methylation in p16 promoter was found in 100% of tumors, but the pattern of methylation varied depending on tumor type. The incidence and extent of de novo methylation in the CpG island of the p16 promoter increased with tumor progression. To further explore the evolution of p16 gene hypermethylation, we examined the appearance and progression of site-specific de novo methylation during early preneoplasia. Our data show that site-specific de novo methylation of 5' CpG island of p16 gene precedes tumor development and undergoes dynamic expansion during tumor progression.