Heart cells contain ATP-sensitive potassium (K_ATP) channels in both the sarcolemma and the inner mitochondrial membrane. The sarcolemmal channels are believed to be heteromultimeric complexes of sulfonylurea receptors (SUR) and potassium inward rectifier (Kir) gene products, but the molecular identity of mitochondrial K_ATP(mitoK_ATP) channels remains unclear. To probe the molecular composition of K_ATPchannels, we used adenoviral gene transfer to express wild-type (WT) and dominant-negative (AFA) constructs of Kir6.1 and 6.2 in rabbit ventricular myocytes. None of the Kir6.1 or 6.2 constructs affected mitoK_ATPchannel activity as assayed by confocal imaging of flavoprotein fluorescence, contradicting the proposal, based on subcellular antibody localization, that Kir6.1 forms part of mitoK_ATPchannels. As previously reported, dominant-negative Kir6.2 gene transfer suppressed sarcolemmal K_ATPcurrent, while Kir6.1 constructs had no effect on sarcolemmal activity. Immunohistochemistry with an anti-Kir6.1 antibody revealed expression of this protein in heart but no apparent co-localization with mitochondria. Thus, the available evidence indicates that both Kir6.1 and 6.2 are expressed in ventricular myocytes, but neither plays a discernible functional role in the mitoK_ATPchannel.