Abnormalities in the ret proto-oncogene are found in several disorders such as multiple endocrine neoplasia (MEN) 2, sporadic medullary thyroid cancer, congenital megacolon and papillary thyroid cancer. In MEN 2A or 2B, early genetic diagnosis before the development of clinical tumors is crucial for the cure of the disease. We studied mutations of ret proto-oncogene in 3 Korean families with MEN 2A and searched for new restriction sites that could be used for genetic diagnosis. By direct sequencing of exon 10 and 11 harboring 'hot' spots, heterozygous point mutation was detected at positions translating cysteine codon in all 3 families. In 2 families, mutations at codon 634 in exon 11 were found (from TGC to CGC or TAC), yielding a new CfoI or RsaI restriction site. In one family, a mutation was located at codon 618 in exon 10 (from TGC to CGC), generating a new CfoI restriction site. These new restriction sites were used in detecting 2 undiagnosed family members without clinical symptoms or signs. In one of them, thyroidectomy was performed to disclose a small medullary thyroid cancer. These results indicate that Korean MEN 2A patients have germ-line mutations in the ret protooncogene at the cysteine residues like patients of other races, and the strategy employing direct sequencing to find mutations at 'hot spot' and search for ensuing new restriction sites could be a useful approach for the molecular diagnosis of genetic diseases accompanied by mutations in restricted areas of disease genes such as MEN 2.