We have investigated the subunit stoichiometry of the pancreatic beta-cell ATP-sensitive K+ (KATP) channel (SUR1/Kir6.2 channel) by constructing cDNA encoding a single polypeptide (beta alpha polypeptide) consisting of a SUR1 (beta) subunit and a Kir6.2 (alpha) subunit. 86Rb+ efflux and single-channel properties of COS1 cells expressing beta alpha polypeptides were similar to those of COS1 cells coexpressing alpha monomers and beta monomers. Coexpression of beta alpha polypeptides with alpha monomers inhibited the K+ currents, while coexpression with beta monomers did not. We then constructed another single polypeptide (beta alpha2) consisting of a beta subunit and a dimeric repeat of the alpha subunit. 86Rb+ efflux from COS1 cells expressing beta alpha2 polypeptides was small, but was restored by supplementation with beta monomers. These results indicate that the activity of K(ATP) channels is optimized when the alpha and beta subunits are coexpressed with a molar ratio of 1:1. Since inward rectifier K+ channels are thought to function as homo- or hetero-tetramers, this suggests that the K(ATP) channel functions as a multimeric protein, most likely a hetero-octamer composed of a tetramer of the Kir6.2 subunit and a tetramer of the SUR1 subunit.