Despite extensive study since the first report of familial benign hypercalcemia (FBH, or hypocalciuric hypercalcemia) in 1972, there is no evidence of the specific abnormal gene product. FBH is highly suitable for either a candidate gene or a reverse genetics approach to localizing the genetic abnormality, because it is inherited in an autosomal dominant pattern, is highly penetrant, does not affect survival, and can be diagnosed in families with readily available measurements. Importantly, several candidate genes have been cloned and mapped. Therefore, we collected blood samples and extracted leukocyte DNA from 94 members of 4 families with well documented FBH (44 affected, 45 unaffected, and 5 unclassifiable). We digested the DNA samples with various restriction endonucleases, conducted standard Southern blotting, and searched for restriction fragment length polymorphisms for the following candidate genes (probe names in parentheses): multiple endocrine neoplasia (MEN) type 1 (pMCMP.1, pHBI59, p3C7, and pTHH26), MEN 2a (MCK2 and cTB14.34), basic fibroblast growth factor (pHFL1-7), (Ca2+,Mg2+)ATPase isoform 4 (hPMCA4), membrane Na/Ca exchanger (cNC28 M-A), PTH (pPTH-LF), and calbindin-D28K (pSKCalb). In addition, we used the anonymous variable number tandem repeat marker pYNH24 to verify pedigree structures by excluding misinheritances. Data were analyzed using the Linkage program. For none of the genes was there significant linkage with the FBH trait; logarithm of odds scores ranged from -1.3 to -26.0 at a recombination fraction of 0.001, and from 0.6 to -5.6 at a recombination fraction of 0.10. We conclude that FBH is unrelated to the MEN syndromes and is not caused by mutations in any of the calcium-regulating or -binding proteins or growth factors studied thus far.