Cardiac sarcolemmal ATP-sensitive potassium (sarcoKATP) channels have been generally considered to comprise Kir6.2/SUR2A subunits in an octomeric complex. These channels, inhibited by normal physiological ATP, are activated by ATP depletion and concomitant ADP increases to reduce electrical excitability to preserve ATP. SarcoKATP opening during metabolic stress, may reduce infarct size and aid recovery by shortening the cardiac action potential duration so decreasing Ca2+ influx. Kir6.1 subunits have been suggested to comprise part of the mitochondrial KATP channel complex and are hypothesised to be involved in cardioprotection. Here we present data demonstrating expression of Kir6.1-containing channels at the sarcolemmal membrane. This Kir6.1-like channel can be inhibited by the Kir6.1 pore blocking compound PNU-37883A (1) and also using shRNA knockdown of Kir6.1.Cardiomyocytes were isolated by enzymatic digestion using Langendorff apparatus from excised hearts from adult male Wistar rats. Cell-attached patch recording was used to assess the single channel currents on the cardiomyocyte sarcolemma. Cardiomyocytes were perfused with normal Tyrode’s (NT) solution at 30±2 °C and currents were recorded at a holding potential equivalent to -110 mV. The Kir6.1-like channel amplitude at -110 mV was 5.4±0.3 pA (n=9) compared to Kir6.2-containing channels, activated by metabolic inhibition with cyanide and iodoacetic acid, which had a single channel current of 9.8±0.2 pA (n=6). Using current-voltage measurements the single channel conductance was calculated to be 39.1±0.4pS in Kir6.1-like channels compared to 83.4±1.1 pS in Kir6.2 containing channels. Interestingly, unlike Kir6.2, Kir6.1 openings occurred continuously with an open probability (Po) of 0.12±0.04 (n=11). When cardiomyocytes were treated with 3μM PNU-37883A in the perfusate, the Po was significantly reduced (0.007±0.001, n=6, P<0.05). Knockdown of Kir6.1 expression, by adenoviral infection with shRNA, significantly inhibited the Kir6.1 channel current. After 24h transfection channel Po was decreased to 0.008±0.002 (n=10, P<0.01) compared to 24h cultured control group (0.10±0.02, n=6). All data was compared using Students t-test. These data demonstrate that a Kir6.1-like conductance is expressed at the sarcolemmal surface of rat cardiomyocytes. This current can be inhibited by a Kir6.1-pore blocker and can also be reduced by gene knockdown. These findings suggest that both Kir6.1 and Kir6.2 pore-forming subunits functionally express at the cell surface where the relatively ATP-insensitive Kir6.1 is open for a large proportion of the time. This channel may play a role in the regulation of membrane potential and action potential duration