The ATP-sensitive K-channel plays a central role in insulin release from pancreatic beta cells. This channel consists of two subunits: a sulphonyl-urea receptor, SUR1, and an inwardly rectifying K-channel subunit, Kir6.2. We screened 135 white Caucasian patients with non-insulin-dependent diabetes mellitus (NIDDM) and 90 non-diabetic subjects for mutations in the Kir6.2 gene by single-stranded conformational polymorphism (SSCP) analysis. We identified one silent mutation (A190A) and four missense mutations (E23K, L270V, I337V and S385C) in normal and diabetic individuals. In a single diabetic subject, we identified a two-amino acid insertion (380KP). We also screened 39 Afro-Caribbean diabetic subjects and identified one additional missense (L355P) and one more silent (S363S) mutation. The E23K and I337V variants were completely linked. The common variants (E23K, 1337V and L270V) were found with similar frequency in diabetic and normal subjects. Diabetic subjects with the variants responded normally to sulphonylurea therapy. When mutant Kir6.2 subunits were coexpressed with SUR1 in Xenopus oocytes, there was no difference in the sensitivity of the whole-cell currents to metabolic inhibition or to the sulphonylurea tolbutamide. We therefore conclude that mutations in Kir6.2 are unlikely to be a major cause of NIDDM.