1. The outermost charged amino acid of S4 segments in the alpha subunit of human skeletal muscle sodium channels was mutated to cysteine in domains I (R219C), II (R669C), III (K1126C), and IV (R1448C). Double mutations in DIS4 and DIVS4 (R219C/R1448C), DIIS4 and DIVS4 (R669C/R1448C), and DIIIS4 and DIVS4 (K1126C/R1448C) were introduced in other constructs. Macropatch recordings of mutant and wild-type (hSkM1-wt) skeletal muscle sodium channels expressed in Xenopus oocytes were used to measure deactivation kinetics from open or fast inactivated states. 2. Conductance (voltage) curves (G (V)) derived from current (voltage) (I (V)) relations indicated a right-shifted G (V) relationship for R669C and for R669C/R1448C, but not for other mutations. The apparent valency was decreased for all mutations. Time-to-peak activation at -20 mV was increased for R1448C and for double mutations. 3. Deactivation kinetics from the open state were determined from the monoexponential decay of tail currents. Outermost charge-to-cysteine mutations in the S4 segments of domains III and IV slowed deactivation, with the greatest effect produced by R1448C. The deactivation rate constant was slowed to a greater extent for the DIII/DIV double mutation than that calculated from additive effects of single mutations in each of these two domains. Mutation in DIIS4 accelerated deactivation from the open state, whereas mutation in DIS4 had little effect. 4. Delays in the onset to recovery from fast inactivation were determined to assess deactivation kinetics from the inactivated state. Delay times for R219C and R669C were not significantly different from those for hSkM1-wt. Recovery delay was increased for K1126C, and was accelerated for R1448C. 5. Homologous charge mutations of S4 segments produced domain-specific effects on deactivation gating from the open and from the fast inactivated state. These results are consistent with the hypothesis that translocations of S4 segments in each domain during deactivation are not identical and independent processes. Non-identical effects of these mutations raise several possibilities regarding deactivation gating; translocation of DIVS4 may constitute the rate-limiting step in deactivation from the open state, DIVS4 may be part of the immobilizable charge, and S4 translocations underlying deactivation in human skeletal muscle sodium channel may exhibit co-operativity.