Closure of ATP-sensitive potassium channels in pancreatic islet β-cells initiates a cascade of events that leads to insulin secretion. β-Cell ATP-sensitive potassium currents can be reconstituted by coexpression of the inward rectifier Kir6.2 and the sulfonylurea receptor (SUR), a member of the ATP-binding cassette superfamily. Mutations in SUR have been identified in individuals affected with familial persistent hyper-insulinemic hypoglycemia of infancy (PHHI), an autosomal recessive disorder of glucose metabolism which is linked to chromosome 11p15.1 and characterized by unregulated secretion of insulin and profound hypo-glycemia. Because the Kir6.2 locus is within 5 kilobases (kb) of the SUR gene on chromosome 11p15.1 and it is a necessary member of the β-cell K_ATP channel, we considered Kir6.2 as a candidate gene for PHHI. We identified a homozygous point mutation in Kir6.2 in the genomic DNA of a child, severely affected with PHHI, from a consanguineous family. This mutation is predicted to disrupt the conservedα-helical second transmembrane (M2) domain of the inward rectifier by substitution of a proline for a leucine residue (L147P). Mutation of Kir6.2, like SUR, appears to lead to the PHHI phenotype suggesting that Kir6.2 is necessary, although not sufficient, for normal regulation of insulin release.