Direct DNA analysis permits accurate identification of gene carriers in kindred members with multiple endocrine neoplasia type 2A (MEN 2A). The aim of this study was to assess the specificity of basal and pentagastrin stimulated calcitonin levels in 3 family members with MEN 2A. For this purpose 53 members of 3 consecutive families with MEN 2A were evaluated in a university medical center. Serum calcitonin, basal and stimulated, was determined by a commercial RIA. RET protooncogene analysis was carried out by automatic DNA sequencing and adequate digestion of PCR amplified products for exons 10 and 11. Two distinct mutations in the RET protooncogene were identified. A T-->A transition at position 1783 (codon 618) in exon 10 was detected in one family, and a G-->A replacement at position 1832 (codon 634) in exon 11 in the others. In non-gene carriers we obtained 6.6% of false-positive results for basal calcitonin and 15.4% for the pentagastrin provocative test. We conclude that the specificity of basal and pentagastrin-stimulated calcitonin is rather limited and RET protooncogene analysis must be the first line screening procedure in order to identify gene carriers.