Glibenclamide Block of KATPChannels.Introduction:The mechanism by which glibenclamide inhibits KATPchannel activity has been examined in membrane patches from isolated rat ventricular cells.Methods and Results:Inside‐out patches were exposed to zero, or low, [ATP] to activate KATPchannels. Glibenclamide did not affect single channel conductance, but reversibly reduced channel open probability from either side of the membrane. Internal (cytoplasmic) glibenclamide inhibited with half‐maximal inhibitory [glibenclamide] = 6 μM, Hill coefficient = 0.35. Complete channel inhibition was not observed, even at 300μM[glibenclamide]. The response to step increases of internal [glibenclamide]could be resolved into two phases of channel inhibition (t1/2, fast,<1 sec, t1/2, slow= 10.5 ± 0.9 sec, n = 8). Step decrease of [glibenclamide] caused a single resolvable phase of reactivation (t1/2= 20.4 ± 0.7 sec, n = 16). Channel inhibition by internal glibenclamide could be relieved by ADP, but only in the presence of Mg2+.Conclusion:Glibenclamide can inhibit KATPchannels from either side of the membrane, with block from one side being competitive with block from the other. Internal MgADP antagonizes the blocking action of glibenclamide. Glibenclamide inhibition of cardiac KATPchannels differs quantitatively and qualitatively from the inhibition of pancreatic KATPc