A cDNA clone encoding an inwardly-rectifying potassium channel subunit (Kir6.2) was isolated from an insulinoma cDNA library. The mRNA is strongly expressed in brain, skeletal muscle, cardiac muscle and in insulinoma cells, weakly expressed in lung and kidney and not detectable in spleen, liver or testis. Heterologous expression of Kir6.2 in HEK293 cells was only observed when the cDNA was cotransfected with that of the sulphonylurea receptor (SUR). Whole-cell Kir6.2/SUR currents were K(+)-selective, time-independent and showed weak inward rectification. They were blocked by external barium (5 mM), tolbutamide (Kd = 4.5 microM) or quinine (20 microM) and by 5 mM intracellular ATP. The single-channel conductance was 73 pS. Single-channel activity was voltage-independent and was blocked by 1 mM intracellular ATP or 0.5 mM tolbutamide. We conclude that the Kir6.2/SUR channel complex comprises the ATP-sensitive K-channel.