Although ATP-sensitive K+ (KATP) channel openers depress force, channel blockers have no effect. Furthermore, the effects of channel openers on single action potentials are quite small. These facts raise questions as to whether 1) channel openers reduce force via an activation of KATP channels or via some nonspecific effects and 2) the reduction in force by KATP channels operates by changes in amplitude and duration of the action potential. To answer the first question we tested the hypothesis that pinacidil, a channel opener, does not affect force during fatigue in muscles of Kir6.2-/- mice that have no cell membrane KATP channel activity. When wild-type extensor digitorum longus (EDL) and soleus muscles were stimulated to fatigue with one tetanus per second, pinacidil increased the rate at which force decreased, prevented a rise in resting tension, and improved force recovery. Pinacidil had none of these effects in Kir6.2-/- muscles. To answer the second question, we tested the hypothesis that the effects of KATP channels on membrane excitability are greater during action potential trains than on single action potentials, especially during metabolic stress such as fatigue. During fatigue, M wave areas of control soleus remained constant for 90 s, suggesting no change in action potential amplitude for half of the fatigue period. In the presence of pinacidil, the decrease in M wave areas became significant within 30 s, during which time the rate of fatigue also became significantly faster compared with control muscles. It is therefore concluded that, once activated, KATP channels depress force and that this depression involves a reduction in action potential amplitude.