Sarcolemmal K_ATP channels are activated after pathological depletion of intracellular ATP, unlike their pancreatic -cell counterparts which dynamically regulate membrane excitability in response to changes in blood glucose. We have recently engineered a series of transgenic (TG) mice overexpressing an ATP-insensitive Kir6.2 mutant (Kir6.2[N30,K185Q]) or the accessory SUR2A (FLAG-SUR2A) or SUR1 (FLAG-SUR1) subunits of the K_ATP channel, under transcriptional control of the MHC promoter. In the present study, we generated double transgenic (DTG) animals overexpressing both Kir6.2[N30,K185Q] and FLAG-SUR1 or FLAG-SUR2A, and examined the effects on cardiac excitability in vivo. No animals expressing both FLAG-SUR1 and Kir6.2[N30,K185Q] transgenes at a high level were obtained. DTG mice expressing one transgene at a high level and the other at a lower level are born, but die prematurely. Electrocardiographic analysis of both anesthetized and conscious animals reveals a constellation of arrhythmias in DTG animals, but not in wild type or single TG littermates. The proarrhythmic effect of the transgene combination is intrinsic to the myocardium, since it persists in isolated hearts. Importantly, this effect is specific for SUR1-expressing DTG animals: DTG animals expressing both Kir6.2[N30,K185Q] and FLAG-SUR2A at high level exhibit neither impaired survival nor increased arrhythmia frequency, even with both subunits expressed at high levels. In demonstrating the profound arrhythmic consequences of K_ATP channels comprised of SUR1 and Kir6.2[N30,K185Q] in the myocardium, specifically, the results highlight the critical differential activation of SUR1 versus SUR2A, and indicate that expression of hyperactive K_ATP in the heart is likely to be proarrhythmic.