Glibenclamide, a blocker of ATP-sensitive K+ (KATP) channels, was tested on three different types of rat skeletal K+ channels incorporated into bilayers. Glibenclamide (10 microM) blocked a class of KATP channels (unitary conductance of 57 pS in symmetric 150 mM KCl), which were inhibited by ATP. High concentrations of glibenclamide (100 microM) had no effect on either voltage-gated K+ channels (37 pS), or Ca(2+)-activated K+ channels (210 pS). Our results show that glibenclamide, even at high concentrations (100 microM) that may be required for quick action in whole muscle experiments, is a selective and specific blocker of skeletal KATP channels.