BACKGROUND: There is increasing interest in DNA methylation and in its implication in transcriptional gene silencing, a phenomenon commonly seen in human cancer. AIMS: To develop a new method that would allow quantitative DNA methylation analysis in a large range of clinical samples, independently of the processing protocol. METHODS: A methylation sensitive dot blot assay (MS-DBA) was developed, which is quantitative and combines bisulfite modification, PCR amplification using primers without CpG sites, and dot blot analysis with two probes specific for methylated and unmethylated DNA. RESULTS: The established method was used to study methylation of the hTERT, APC, and p16 promoter regions in microdissected, formalin fixed and paraffin wax embedded tissues. CONCLUSIONS: MS-DBA is a sensitive, specific, and quantitative approach to analyse DNA methylation in a variety of frozen or fixed tissues. Moreover, MS-DBA is rapid, easy to perform, and permits the screening of a large panel of samples in one experiment. Thus, MS-DBA can facilitate the routine analysis of DNA methylation in all types of clinical samples