The methylation of MspI/HpaII sites flanking a variable tandem repeat in the 3' region of the c-Ha-ras protooncogene was analyzed in 74 DNA samples of non-small cell lung carcinomas and control lung tissues. Of 39 informative samples, 7 allelic deletions (18%) were found at the c-Ha-ras locus and of these, five (71%) showed hypomethylation of the nondeleted allele. Heterozygous DNA samples without allele loss revealed hypomethylation in 37% (12 of 32). Among 35 homozygotes, 9 showed hypomethylation (26%). We also analyzed c-Ha-ras mutations at codons 12, 13, and 61 by polymerase chain reaction and designed restriction fragment length polymorphism and found no mutation. Thus, c-Ha-ras mutations are not associated with the development of the detected abnormalities. We conclude that hypomethylation at specific sites in the 3' region is associated with loss of heterozygosity for the c-Ha-ras gene in non-small cell lung cancer. The finding that, in informative samples, hypomethylation occurs 2-3 times more frequently than allelic loss suggests that it might be a change contributing or predisposing to a genetic instability that can ultimately lead to c-Ha-ras allelic deletions found in tumor DNA.