1. The role of K+ channels and membrane potential in myoblast fusion was evaluated by examining resting membrane potential and timing of expression of K+ currents at three stages of differentiation of human myogenic cells: undifferentiated myoblasts, fusion-competent myoblasts (FCMBs), and freshly formed myotubes. 2. Two K+ currents contribute to a hyperpolarization of myoblasts prior to fusion: IK(NI), a non-inactivating delayed rectifier, and IK(IR), an inward rectifier. 3. IK(NI) density is low in undifferentiated myoblasts, increases in FCMBs and declines in myotubes. On the other hand, IK(IR) is expressed in 28% of the FCMBs and in all myotubes. 4. IK(IR) is reversibly blocked by Ba2+ or Cs+. 5. Cells expressing IK(IR) have resting membrane potentials of -65 mV. A block by Ba2+ or Cs+ induces a depolarization to a voltage determined by IK(NI) (-32 mV). 6. Cs+ and Ba2+ ions reduce myoblast fusion. 7. It is hypothesized that the IK(IR)-mediated hyperpolarization allows FCMBs to recruit Na+, K+ and T-type Ca2+ channels which are present in these cells and would otherwise be inactivated. FCMBs, rendered thereby capable of firing action potentials, could amplify depolarizing signals and may accelerate fusion.