Background The sarcolemmal adenosine triphosphate-sensitive potassium channel (sKATP), composed primarily of potassium inward rectifier (Kir) 6.2 and sulfonylurea receptor 2A subunits, has been implicated in cardiac myocyte volume regulation during stress; however, it is not involved in cardioprotection by the adenosine triphosphate-sensitive potassium channel (KATP) channel opener diazoxide (7-chloro-3-methyl-1,2,4-benzothiadiazine-1,1-dioxide [DZX]). Paradoxically, the sKATP channel subunit Kir6.2 is necessary for detrimental myocyte swelling secondary to stress. The Kir6.1 subunit can also contribute to KATP channels in the heart, and we hypothesized that this subunit might play a role in myocyte volume regulation in response to stress. Study Design Isolated cardiac myocytes from either wild-type mice or mice lacking the Kir6.1 subunit (Kir6.1[-/-]) were exposed to control Tyrode's solution (TYR) for 20 minutes, test solution (TYR, hypothermic hyperkalemic cardioplegia [CPG], or CPG + KATP channel opener DZX [CPG + DZX]) for 20 minutes, followed by TYR for 20 minutes. Myocyte volume and contractility were measured and analyzed. Results Both wild-type and Kir6.1(-/-) myocytes demonstrated substantial swelling during exposure to stress (CPG), which was prevented by DZX. Wild-type myocytes also demonstrated reduced contractility during stress of CPG that was prevented by DZX. However, Kir6.1(-/-) myocytes did not show reduced contractility during stress. Conclusions These data indicate that KATP channel subunit Kir6.1 is not necessary for DZX's maintenance of cell volume during the stress of CPG. The absence of Kir6.1 does not affect the contractile properties of myocytes during stress, suggesting the absence of Kir6.1 improves myocyte tolerance to stress via an unknown mechanism.