Many protocols for gene-specific DNA methylation analysis are either labor intensive, not quantitative and/or limited to the measurement of the methylation status of only one or very few CpG positions. Pyrosequencing is a real-time sequencing technology that overcomes these limitations. After bisulfite modification of genomic DNA, a region of interest is amplified by PCR with one of the two primers being biotinylated. The PCR generated template is rendered single-stranded and a pyrosequencing primer is annealed to analyze quantitatively cytosine methylation. In comparative studies, pyrosequencing has been shown to be among the most accurate and reproducible technologies for locus-specific DNA methylation analyses and has become a widely used tool for the validation of DNA methylation changes identified in genome-wide studies as well as for locus-specific analyses with clinical impact such as methylation analysis of the MGMT promoter. Advantages of the Pyrosequencing technology are the ease of its implementation, the high quality and the quantitative nature of the results, and its ability to identify differentially methylated positions in close proximity.