Skeletal muscle explants from normal subjects were established from biopsy material on collagen. Cellular outgrowth appeared within 3–4 days, and fusion of myoblasts was observed in 5–10 days. Multinucleated myotubes were impaled under high optical magnification, at 37°C, with conventional glass microelectrodes. The mean resting potential was −44.4 mV ± 2.4 (n = 399); −33 ± 2.3 mV at 9 days (n = 10) vs −48 ± 2.5 mV (n = 15) at 27 days. The average input resistance (R_in) was 9.7 M ω (n = 83). Action potentials could be elicited by electrical stimulation and had a mean amplitude of 55.9 ± 2.1 mV with a mean maximum rate of rise (±V_max) of 72.1 ± 7.5 V/s. The mean overshoot was 13.9 ± 2.3 mV, and the action potential duration determined at 50% of repolarization (APD₅₀) was 8.0 msec (n = 7). The resting membrane potential showed a depolarization of 23 mV/decade for extracellular potassium ion concentration ([K]_o) between 4.5–100 mM. Thus, we have established the normal resting potential and maximum rate of rise of the action potential for human myotubes in culture. We have shown that the values for these are less than those previously reported in cultured avian and rodent cells. In addition, we have shown that the response in our system of the resting potential to change in extracellular potassium concentration is blunted compared to studies using isolated muscle, suggesting an increase in ratio of sodium to potassium permeability. Cultured human muscle cells depolarized in the presence of ouabain.