Mapping of genomic DNA methylation is a dispensable part of functional genome. We have developed a novel method based on methylation-specific primer and serial analysis of gene expression, called MSP-SAGE with potential of high-throughput quantification of genomic DNA methylation. We used a 6-mer methylation-specific primer to extend the methylated CpG sequences other than nonmethylated CpG sequences. The 17 bp tags contained methylated CpG sequence, which were obtained from extended methylation sequence by digestion of restriction endonuclease, and then the tags were concatenated and cloned for sequencing. We can identify the locations of methylation according to the sequences of tags and quantity the methylation status from the frequency of the tags. MSP-SAGE has a good linearity in a broad rnethylation range from 5% to 100% with good accuracy and high precision. The proof-of-principle study shows that MSP-SAGE is a reliable high-throughput assay for quantification of DNA methylation. (c) 2006 Elsevier Inc. All rights reserved.