To assess the pathophysiological role of the RET protooncogene in sporadic pheochromocytomas, we examined the 2 regions of the gene in which molecular defects are specifically associated with the multiple endocrine neoplasias (MEN) type 2A (the cysteine-rich domain encoded by exons 10 and 11), and type 2B (the tyrosine kinase domain encoded by exon 16). The sequences of both regions were amplified by reverse transcriptase-polymerase chain reaction (PCR) or PCR from tumor RNA and/or leukocyte DNA. The amplified fragments were analyzed by denaturing gradient gel electrophoresis using chemical clamps. In 28 patients with unilateral sporadic tumors, 6 RET mutations were found, 3 in the MEN 2A region, 3 in the MEN 2B region. Five patients had missense mutations: 2 in the MEN 2A region (C634W and D631Y), and 3 in the MEN 2B region (M918T). Analysis of leukocyte DNA in 3 of these patients confirmed that RET mutations were only present in tumor DNA. The sixth patient had lost exon 10 in the tumor complementary DNA as a result of the deletion of the dinucleotide -AG- at the 3'splice acceptor site of intron 9; this molecular defect was only found in the tumor DNA. Thus RET mutations of the MEN 2A and 2B regions are also found in about 20% of sporadic pheochromocytomas. We describe new types of molecular defects of the RET protooncogene in the MEN 2A region that involve noncysteine residues and loss of exon 10. Further studies should be extended to analyze the entire RET protooncogene. These findings have a profound clinical impact for the management of patients with supposedly sporadic pheochromocytomas.