Site-specific methylation of the human c-myc oncogene was investigated in a set of cultured human tumor cell lines and normal human fibroblast strains. As previously reported, all of the tumor cell lines in contrast to normal cells were hypomethylated to various degrees in their total genomic DNA. The presence of methylation in specific regions of the c-myc gene was analyzed by use of the restriction endonuclease isoschizomers MspI and HpaII, which recognize the sequence 5'-CCGG-3' (CCGG = DNA sequence of 2 cytosine bases followed by 2 guanine bases) but differ in their abilities to cleave at the internal cytosine residue when it is methylated. The first exon, first intervening sequence, and the second exon were hypomethylated in all cell types, regardless of whether the cells were normal or oncogenically transformed and regardless of the degree of total genomic methylation of the cell. However, the solitary CCGG site in the third exon was fully methylated in normal cell strains. In contrast, in 3 of 5 tumor cell lines measured, this site was hypomethylated. This is the first demonstration of a site-specific DNA methylation defect in a cellular oncogene. Some possible implications relating disruption of DNA methylation to oncogene control and oncogenesis are discussed.