Hirschsprung disease (HSCR), or congenital aganglionic megacolon, is the most common cause of congenital bowel obstruction with an incidence of 1 in 5000 live births. Recently, linkage of an incompletely penetrant, dominant form of HSCR was reported, followed by identification of mutations in the RET receptor tyrosine kinase. To determine the frequency of RET mutations in HSCR and correlate genotype with phenotype, we have screened for mutations among 80 HSCR probands representing a wide range of phenotypes and family structures. Polymerase chain reaction (PCR) and single-strand conformation polymorphism (SSCP) analysis of RET's 20 exons for mutations among probands revealed eight putative mutations (10%). Sequence changes, which included missense, frameshift and complex mutations, were detected in both familial and isolated cases, among patients with both long- and short-segment HSCR and in three kindreds with other phenotypes (maternal deafness, talipes and malrotation of the gut, respectively). Two mutations (C609Y and C620R) we identified have previously been associated with multiple endocrine neoplasia type 2A (MEN2A), medullary thyroid carcinoma (MTC) and, on rare occasions, HSCR. Thus, while HSCR family members may be at risk for developing neuroendocrine tumors, it follows that identical mutations in RET may be able to participate in the pathogenesis of distinct phenotypes. Our data suggest that: (i) the overall frequency of RET mutations in HSCR patients is low and therefore, other genetic and/or environmental determinants contribute to the majority of HSCR susceptibility, and (ii) at present, there is no obvious relationship between RET genotype and HSCR phenotype.