Classically, cardiac sarcolemmal K_ATP channels have been thought to be composed of Kir6.2 (KCNJ11) and SUR2A (ABCC9) subunits. However, the evidence is strong that SUR1 (sulfonylurea receptor type 1, ABCC8) subunits are also expressed in the heart and that they play a significant functional role in the atria. To examine this further, we have assessed the effects of isotype-specific potassium channel-opening drugs, diazoxide (specific to SUR1>SUR2A) and pinacidil (SUR2A>SUR1), in intact hearts from wild-type mice (WT, n=6), SUR1^−/− (n=6), and Kir6.2^−/− mice (n=5). Action potential durations (APDs) in both atria and ventricles were estimated by optical mapping of the posterior surface of Langendorff-perfused hearts. To confirm the atrial effect of both openers, isolated atrial preparations were mapped in both WT (n=4) and SUR1^−/− (n=3) mice. The glass microelectrode technique was also used to validate optical action potentials. In WT hearts, diazoxide (300 μM) decreased APD in atria (from 33.8±1.9 ms to 24.2±1.1 ms, p<0.001) but was without effect in ventricles (APD 60.0±7.6 ms vs. 60.8±7.5 ms, respectively, NS), consistent with an atrial-specific role for SUR1. The absence of SUR1 resulted in loss of efficacy of diazoxide in SUR1^−/− atria (APD 36.8±1.9 ms vs. 36.8±2.8 ms, respectively, NS). In contrast, pinacidil (300 μM) significantly decreased ventricular APD in both WT and SUR1^−/− hearts (from 60.0±7.6 ms to 29.8±3.5 ms in WT, p<0.001, and from 63.5±2.1 ms to 24.8±3.8 ms in SUR1^−/−, p<0.001), but did not decrease atrial APD in either WT or SUR1^−/− hearts. Glibenclamide (10 μM) reversed the effect of pinacidil in ventricles and restored APD to control values. The absence of Kir6.2 subunits in Kir6.2^−/− hearts resulted in loss of efficacy of both openers (APD 47.2±2.2 ms vs. 47.6±2.1 ms and 50.8±2.4 ms, and 90.6±5.7 ms vs. 93.2±6.5 ms and 117.3±6.4 ms, for atria and ventricle in control versus diazoxide and pinacidil, respectively). Collectively, these results indicate that in the same mouse heart, significant differential K_ATP pharmacology in atria and ventricles, resulting from SUR1 predominance in forming the atrial channel, leads to differential effects of potassium channel openers on APD in the two chambers.