ATP-sensitive potassium (KATP) channels have been implicated in the mechanism of cardioprotection imparted by brief periods of non-lethal ischaemia prior to a prolonged insult. Such ischaemic preconditioning (IPC) has been demonstrated to markedly reduce infarct size so improving the recovery after myocardial infarction. After cardioprotective stimuli, sarcolemmal KATP (SarcoKATP) channels are hypothesised to open early during ischaemia to shorten the cardiac action potential (AP), limit calcium entry and so preserve ATP. We present data showing that sarcoKATP channel opening, and contractile failure, is actually delayed after IPC, direct PKC activation or adenosine pre-treatment. Cardiomyocytes were enzymatically isolated from adult male Wistar rats culled in accordance with Home Office regulations. IPC cardiomyocytes were isolated from hearts after an IPC protocol of 3 cycles of 5 minutes of stopped perfusion. Cardioprotection was assessed using a contractile function protocol. Briefly, cardiomyocytes were perfused with normal Tyrode (NT) solution at 32±2°C with 1 Hz electric field stimulation to trigger contractions. Solution was exchanged for a metabolic inhibition (MI) solution (2 mM cyanide and 1 mM iodoacetic acid in substrate free Tyrode solution) for 7 mins, followed by 10 mins of simulated reperfusion with NT solution. The times to contractile failure and the percentage of cells showing contractile function and hypercontracted cells (recorded as a measure of reperfusion injury) were recorded in control, IPC and cells treated with either 1 μM PMA or 3 μM adenosine for 5 mins prior to the contractile function protocol. Cell attached patch recording was used to assess the time to opening of SarcoKATP channels in the presence of MI-Tyrode solution. The opening of SarcoKATP channels was measured as the time to the first burst greater than 500 ms in duration. Using this assessment, SarcoKATP channel opening was significantly delayed in IPC (182±11s***), and PMA (162±6s*) and adenosine (197±8s***) pre-treated cardiomyocytes compared to control (127±6s) (n=8, 6, 8 and 12 respectively,*P<0.05, ***P<0.001, Students t-test). These data demonstrate that IPC, PMA or adenosine impart a marked cardioprotection to isolated cardiomyocytes. However, the opening of SarcoKATP channels is markedly delayed after cardioprotective stimuli. These findings suggest that early opening of SarcoKATP channels is not directly involved in the cardioprotection afforded by IPC or PKC activation. This does not exclude a role for mitochondrial KATP channe