The isoform-specific structure of the ATP-sensitive potassium (KATP) 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 KATP channel in cardiac ventricular myocytes. Using a novel antibody directed against the COOH terminus of SUR1 (ABCC8), we show that this KATP subunit is also expressed in mouse myocardium and is the dominant SUR isoform in the atrium. This suggests differential sarcolemmal KATP composition in atria and ventricles, and, to test this, KATP currents were measured in isolated atrial and ventricular myocytes from wild-type and SUR1 animals. KATP conductance is essentially abolished in SUR1 atrial myocytes but is normal in SUR1 ventricular myocytes. Furthermore, pharmacological properties of wild-type atrial KATP match closely the properties of heterologously expressed SUR1/Kir6.2 channels, whereas ventricular KATP properties match those of heterologously expressed SUR2A/Kir6.2 channels. Collectively, the data demonstrate a previously unappreciated KATP channel heterogeneity: SUR1 is an essential component of atrial, but not ventricular, KATP 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 KATP pharmacologically, as well as for understanding of the role of diazoxide in preconditioning.