The identification of inherited mutations in the RET protooncogene (RET) associated with multiple endocrine neoplasia type 2A (MEN 2A) has enabled the development of a genetic test to identify asymptomatic carriers of disease. Genomic DNA was extracted from 96 members of an MEN 2A kindred. The polymerase chain reaction was used to amplify the RET exon known to contain the associated mutation. The mutation results in a new restriction endonuclease site and is detected by digestion with the appropriate enzyme. Inheritance of the mutation was verified with a previously developed genetic linkage test. We found that (1) mutations vary among kindreds but are consistently inherited within kindreds, (2) invariable correlation exists between mutation and disease (43 mutations in 43 affected individuals), (3) determination of the genetic status by linkage-based testing was precluded by recombination events and the informativeness of genetic markers, and (4) mutation analysis presymptomatically identified two genetically affected individuals. Direct genetic analysis for mutations in RET circumvents the limitations of linkage-based genetic testing and current biochemical screening assays. This method will be the diagnostic test of choice for the identification of asymptomatic individuals at risk for MEN 2A.