AIM: p16 Methylation frequently occurs in carcinogenesis. While it has been hypothesized that the p16 methylation states are dynamically maintained in cancer cells, direct evidence supporting this hypothesis has not been available until now. METHODS: A fusion cell model was established which reprogrammed the native DNA methylation pattern of the cells. The methylation status of the p16 alleles was then repeatedly quantitatively analyzed in the fusion monoclonal, parental cancer cell lines (p16-completely methylated-AGS and unmethylated-MGC803), and HCT116 non-fusion cell using DHPLC and bisulfite sequencing. Histone methylation was analyzed using chromatin immuno-precipitation (ChIP)-PCR. P16 expression status was determined using immuno-staining and RT-PCR. RESULTS: The methylation status for the majority of the p16 alleles was stably maintained in the fusion monoclonal cells after up to 60 passages. Most importantly, focal de novo methylation, demethylation, and hydroxymethylation were consistently observed within about 27% of the p16 alleles in the fusion monoclones, but not the homozygously methylated or unmethylated parental cells. Furthermore, subclones of the monoclones consistently maintained the same p16 methylation pattern. A similar phenomenon was also observed using the p16 hemi-methylated HCT116 non-fusion cancer cell line. Interestingly, transcription was not observed in p16 alleles that were hydroxymethylated with an antisense-strand-specific pattern. Also, the levels of H3K9 and H3K4 trimethylation in the fusion cells were found to be slightly lower than the parental AGS and MGC803 cells, respectively. CONCLUSION: The present study provides the first direct evidence confirming that the methylation states of p16 CpG islands is not only homeostatically maintained, but also accompanied by a dynamic process of transient focal methylation, demethylation, and hydroxymethylation in cancer cells.