To examine the role of sarcolemmal K_ATP channels in cardiac function, we generated transgenic mice expressing GFP-tagged Kir6.2 subunits with reduced ATP sensitivity under control of the cardiac α-myosin heavy chain promoter. Four founder mice were isolated, and both founders and progeny were all apparently normal and fertile. Electrocardiograms from conscious animals also appeared normal, although mean 24-hour heart rate was approximately 10% lower in transgenic animals compared with littermate controls. In excised membrane patches, K_ATP channels were very insensitive to inhibitory ATP: mean K_1/2 ([ATP] causing half-maximal inhibition) was 2.7 mmol/L in high-expressing line 4 myocytes, compared with 51 μmol/L in littermate control myocytes. Counterintuitively, K_ATP channel density was ≈4-fold lower in transgenic membrane patches than in control. This reduction of total K_ATP conductance was confirmed in whole-cell voltage-clamp conditions, in which K_ATP was activated by metabolic inhibition. K_ATP conductance was not obvious after break-in of either control or transgenic myocytes, and there was no action potential shortening in transgenic myocytes. In marked contrast to the effects of expression of similar transgenes in pancreatic β-cells, these experiments demonstrate a profound tolerance for reduced ATP sensitivity of cardiac K_ATP channels and highlight differential effects of channel activity in the electrical activity of the 2 tissues.