1 We have investigated the e ects of the sulphonylurea, glimepiride, currently used to treat type 2 diabetes, on ATP-sensitive K + (K ATP ) currents of rat cardiac myocytes and on their cloned constituents Kir6.2 and SUR2A expressed in HEK 293 cells. 2 Glimepiride blocked pinacidil-activated whole-cell K ATP currents of cardiac myocytes with an IC 50 of 6.8 nM, comparable to the potency of glibenclamide in these cells. Glimepiride blocked K ATP channels formed by co-expression of Kir6.2/SUR2A subunits in HEK 293 cells in outside-out excised patches with a similar IC 50 of 6.2 nM. 3 Glimepiride was much less e ective at blocking K ATP currents activated by either metabolic inhibition (MI) with CN 7 and iodoacetate or by the K ATP channel opener diazoxide in the presence of inhibitors of F 0 /F 1 -ATPase (oligomycin) and creatine kinase (DNFB). Thus 10 mM glimepiride blocked pinacidil-activated currents by 499%, MI-activated currents by 70% and diazoxideactivated currents by 82%. 4 In inside-out patches from HEK 293 cells expressing the cloned K ATP channel subunits Kir6.2/ SUR2A, increasing the concentration of ADP (1 ± 100 mM), in the presence of 100 nM glimepiride, lead to signi®cant increases in Kir6.2/SUR2A channel activity. However, over the range tested, ADP did not a ect cloned K ATP channel activity in the presence of 100 nM glibenclamide. These results are consistent with the suggestion that ADP reduces glimepiride block of K ATP channels. 5 Our results show that glimepiride is a potent blocker of native cardiac K ATP channels activated by pinacidil and blocks cloned Kir6.2/SUR2A channels activated by ATP depletion with similar potency. However, glimepiride is much less e ective when K ATP channels are activated by MI and this may re¯ect a reduction in glimepiride block by increased intracellular ADP.