Aberrant promoter methylation is an epigenetic loss-of-function mutation analogous to point mutations or deletions. To study global methylation changes in human cancers, we use restriction landmark genomic scanning, a two-dimensional gel electrophoresis technique that allows the assessment of methylation patterns in up to 2,000 CpG islands per gel with methylation-sensitive restriction enzymes (NotI or AscI). Aberrant methylation in primary tumors is identified by comparing tumor profiles to profiles from matching normal DNAs. Loci aberrantly methylated in tumors are easily cloned using arrayed boundary libraries, and subsequent database searches allow us to link the methylation events to genes or expressed sequence tags. Important findings from our studies on cancers of human lung, head and neck; medulloblastomas; and acute myeloid leukemia include the following: (1) Identification of nonrandom, tumor-type-specific methylation events1. (2) Significant overrepresentation of methylated loci on chromosome 11 in acute myeloid leukemia. (3) Identification of six new target genes in lung cancer and ten in acute myeloid leukemia. (4) An increased number of methylation events in metastatic head and neck cancers with overlapping and new targets compared with primary tumors. (5) Methylation in the major breakpoint cluster region for medulloblastomas, suggesting a potential link between genetic instability and DNA methylation. Together these data suggest that the extent of DNA hypermethylation in cancer was previously underestimated and that epigenetic events have an outstanding potential for the identification of new tumor suppressor genes as well as diagnostic, prognostic and therapeutic targets.