We investigated mutations of the ret proto-oncogene in Japanese patients with multiple endocrine neoplasia (MEN) type 2A and type 2B. DNAs from pheochromocytomas and/or medullary thyroid carcinomas (MTCs) of five MEN 2A and three MEN 2B patients were amplified by a polymerase chain reaction (PCR) and analyzed. Tumors of four MEN 2A patients had missense mutations in Cys 634 in the extracellular domain of the ret proto-oncogene. The same mutations were detected in normal tissues of the patients, indicating that the mutations had arisen in the germ line. Using a reverse transcriptase(RT)-PCR, both normal and mutant transcripts of the ret proto-oncogene were detected in a tumor of one patient with MEN 2A mutation. In addition, three MEN 2B patients examined had the same point mutation (ATG-->ACG) at codon 918 in the tyrosine kinase domain of the ret proto-oncogene. Since all mutations identified in this study generated new restriction enzyme sites or eliminated a restriction site, the mutant alleles of affected family members could be readily detected without sequencing.