The isoform-specific structure of the ATP-sensitive potassium (K_ATP) channel endows it with differential fundamental properties, including physiological activation and pharmacology. Numerous studies have convincingly demonstrated that the pore-forming Kir6.2 (KCNJ11) and regulatory SUR2A (ABCC9) subunits are essential elements of the sarcolemmal K_ATP channel in cardiac ventricular myocytes. Using a novel antibody directed against the COOH terminus of SUR1 (ABCC8), we show that this K_ATP subunit is also expressed in mouse myocardium and is the dominant SUR isoform in the atrium. This suggests differential sarcolemmal K_ATP composition in atria and ventricles, and, to test this, K_ATP currents were measured in isolated atrial and ventricular myocytes from wild-type and SUR1^−/− animals. K_ATP conductance is essentially abolished in SUR1^−/− atrial myocytes but is normal in SUR1^−/− ventricular myocytes. Furthermore, pharmacological properties of wild-type atrial K_ATP match closely the properties of heterologously expressed SUR1/Kir6.2 channels, whereas ventricular K_ATP properties match those of heterologously expressed SUR2A/Kir6.2 channels. Collectively, the data demonstrate a previously unappreciated K_ATP channel heterogeneity: SUR1 is an essential component of atrial, but not ventricular, K_ATP channels. Differential molecular make-up of the 2 channels underlies differential pharmacology, with important implications when considering sulfonylurea therapy or dissecting the role of cardiac K_ATP pharmacologically, as well as for understanding of the role of diazoxide in preconditioning.