We have applied a methylation-sensitive restriction endonuclease, Notl, to the existing amplified fragment length polymorphism (AFLP) method and developed Notl-Msel methylation-sensitive-AFLP (MS-AFLP). Notl-Msel MS-AFLP allows the analysis of DNA methylation alterations at the Notl sites scattered over the genome. Hypermethylation and hypomethylation are visualized by the decrease and increase in the band intensity of DNA fingerprints. Identification of consistent changes can be facilitated through parallel electrophoresis of multiple samples. DNA fragments exhibiting alterations can be cloned from fingerprint bands by amplification of gel-eluted DNA with the same pair of primers used for radioactive fingerprint presentation. Fluorescent Notl-Msel MS-AFLP offers a safer method of studying the alterations in DNA methylation, and may be applied to the hybridization of DNA microarrays in the future. Using Notl-Msel MS-AFLP, we observed frequent hypomethylation of a satellite DNA repeat sequence in a majority of breast tumors.