Persistent hyperinsulinemic hypoglycemia of infancy (PHHI), a rare disorder due to defective negative feedback regulation of insulin secretion by low glucose levels, is often familial. Most cases are recessively inherited, and mutations of the sulfonylurea receptor gene (SUR) or the closely linked KIR6.2 gene have been found in several families. Both of these genes encode components of the potassium channels responsible for glucose-regulated insulin release. However, in some families recessive PHHI is not linked to the SUR-KIR6.2 locus, suggesting genetic heterogeneity. We report here a French Canadian kindred with hypoglycemia in five first cousins. All five patients had documented hypoglycemia, and all responded well to diazoxide. In two, inappropriately elevated insulin levels during hypoglycemia were documented. This familial clustering strongly suggests the existence of an autosomal dominant form of PHHI. By preliminary linkage analysis, we tested the possibility of a dominant negative SUR or KIR6.2 mutant. The insulin (INS) and glucokinase (GCK) genes were also tested as additional candidates. Microsatellite markers closely linked to each gene were used, and large negative Lod scores were obtained at the known recombination fractions between all three genes studied and the corresponding marker. We conclude that mutation of a gene other than SUR or KIR6.2 is responsible for the dominant PHHI in this family, and this gene cannot be INS or GCK. We propose that a genome-wide search for this gene is important for elucidating this rare disorder and, more importantly, for determining its potential impact on understanding noninsulin-dependent diabetes mellitus and on the effort to develop bioengineered beta-cells for transplantation.