Objective: Insulin hyperpolarizes skeletal muscle fibers. This has been attributed to electrogenic properties of the Na⁺/K⁺- ATPase (NKA) which is activated by insulin. Low doses of Na⁺-ionophores like amphotericin B (AMB) or monensin do hyperpolarize cells which is also thought to be due to indirect activation of NKA by inreased Na⁺- influx. We have tested the hypothesis, that the hyperpolarization is a consequence of activation of ATP-sensitive K⁺ channels (K_ATP) in both cases. These channels could be activated by NKA's increased ATP utilization. Methods: Muscle preparations were from diaphragms of Wistar rats, continously superfused with a modified Krebs-Ringer-solution (in mM: NaCl 115.5, KCl 3, MgSO4 0.8, Na2HPO4 1, NaHCO3 25, CaCl2 2, glucose 10). pH was adjusted to 7.4 by bubbling with 95% O2 and 5% CO2. Membrane potentials (V_m) were measured with glass microelectrodes having resistances between 5 to 10MW when filled with 3M KCl, tip potentials did not differ between experimental groups. Muscles were treated with amphotericin B (2mM), glibenclamide (4mM), ouabain (1mM), cromakalim (100mM) and insulin (100nM). Control solutions contained equal amounts of solvent. Results: V_m of control muscle fibers were -78.9+/­5.4mV. Insulin and AMB caused hyperpolarizations by 5.8 and 5.9mV, respectively. These effects were statistically significant (p<0.02) and could be completely inhibited by glibenclamide. In the presence of ouabain (V_m=- 76.6±9.4mV) insulin, AMB and glibenclamide had no effect on Vm. Cromakalim caused a depolarization (V_m=- 66.8±12.8mV) of ouabain-treated muscle fibers. Conclusion: Hyperpolarization of skeletal muscle fibers by insulin and AMB is primarily caused by an activation of ATP-sensitive K⁺ channels. The effect depends on an increased subsarcolemmal energy consumption by Na⁺/K⁺-pumps. Therefore, it is likely that channel activation is directly linked to an enhanced ATP utilization.