To develop a quantitative method for methylation analysis of the p16 gene based on mismatch hybridization and chemiluminescence. Methods Genomic DNA was modified by sodium bisulfite to convert all unmethylated but not methylated cytosines to uracil, and subsequently a pair of primer having no CpG sites was designed for amplification target DNA containing methylated or unmethylated CpG sites. The PCR product spanning CpG sites were hybridized with two oligonucleotide probes which perfectly matched the methylated and unmethylated CpG sequences respectively, and the hybrids were detected by chemiluminescent method. The percentage of methylated target sequences could be estimated by calculating the ratio of signals obtained with two probes. Results The percentage of methylation of artificial mixtures DNA showed a linear relation. There was a negative correlation between the methyaltion index with p16 transcriptional mRNA of p16 gene in tumor cell lines. Conclusion Compared with existing methods, this assay is nonisotopic, rapid, simple, and can be widely applied to the study of DNA methylation. To develop a quantitative method for methylation analysis of the p16 gene based on mismatch hybridization and chemiluminescence. Methods Genomic DNA was modified by sodium bisulfite to convert all unmethylated but not methylated cytosines to uracil, and followed by a pair of primer with no CpG sites was designed The PCR product spanning CpG sites were hybridized with two oligonucleotide probes which perfectly matched the methylated and unmethylated CpG sequences respectively, and the hybrids were detected by chemiluminescent method. The percentage of methylated target sequences could be estimated by calculating the ratio of signals obtained with two probes. There was a negative correlation between the methyaltion index with p16 transcriptional mRNA of p16 gene in tumor cell lines. Conclusion The percentage of methylation of artificial mixtures DNA showed a linear relation. with exi sting methods, this assay is nonisotopic, rapid, simple, and can be widely applied to the study of DNA methylation.