The slow inactivation of cloned muscle alpha-subunit Na+ channels was investigated using a Chinese hamster ovary cell line permanently transfected with rat muscle mu1 cDNA. Expression of mu1 Na+ channels was found in cells maintained for more than 6 months after transfection; > 70% of cells expressed >/= 3 nA of Na+ current at +30 mV under whole-cell patch-clamp conditions. As expected, Na+ currents in these cells were blocked by tetrodotoxin as well as by mu-conotoxin. After prolonged depolarization (10 s at +30 mV) to inactivate voltage-gated Na+ channels, Na+ currents slowly reappeared over a time course of several minutes, during which time the cell was repolarized to the holding potential of -100 mV. This recovery from slow inactivation was best fitted by a double exponential function with tau1 = 2.5 s (amplitude = 53%) and tau2 = 83.4 s (amplitude = 38%). In contrast, the development of slow inactivation at +30 mV was best fitted by a single exponential function, with tau = 3.0 s. Steady-state slow inactivation (s infinity) had a midpoint potential (s0.5) of -52 mV and a slope factor (k) of 7.8 mV. Elimination of fast inactivation by treatment with chloramine-T accelerated the development of slow inactivation significantly (by approximately four fold) but had little effect on recovery or on steady-state slow inactivation. Finally, as in cloned brain NaIIA Na+ channels, batrachotoxin abolished both fast and slow inactivation of mu1 Na+ channels. These results together suggest that slow inactivation takes place in the alpha-subunit of mu1 muscle Na+ channels and is governed by a microliter protein region different from that governing fast inactivation.