The steady-state gating of individual batrachotoxin-modified sodium channels in neutral phospholipid bilayers exhibits spontaneous, reversible changes in channel activation, such that the midpoint potential (Va) for the gating curves may change, by 30 mV or more, with or without a change in the apparent gating valence (za). Consequently, estimates for Va and, in particular, za from ensemble-averaged gating curves differ from the average values for Va and za from single-channel gating curves. In addition to these spontaneous variations, the average Va shifts systematically as a function of [NaCl] (being -109, -88, and -75 mV at 0.1, 0.5, and 1.0 M NaCl), with no systematic variation in the average za (approximately 3.7). The [NaCl]-dependent shifts in Va were interpreted in terms of screening of fixed charges near the channels' gating machinery. Estimates for the extracellular and intracellular apparent charge densities (sigma e = -0.7 and sigma i = -0.08 e/nm2) were obtained from experiments in symmetrical and asymmetrical NaCl solutions using the Gouy-Chapman theory. In 0.1 M NaCl the extracellular and intracellular surface potentials are estimated to be -94 and -17 mV, respectively. The intrinsic midpoint potential, corrected for the surface potentials, is thus about -30 mV, and the standard free energy of activation is approximately -12 kJ/mol. In symmetrical 0.1 M NaCl, addition of 0.005 M Ba2+ to the extracellular solution produced a 17-mV depolarizing shift in Va and a slight reduction in za. The shift is consistent with predictions using the Gouy-Chapman theory and the above estimate for sigma e. Subsequent addition of 0.005 M Ba2+ to the intracellular solution produced a approximately 5-mV hyperpolarizing shift in the ensemble-averaged gating curve and reduced za by approximately 1. This Ba(2+)-induced shift is threefold larger than predicted, which together with the reduction in za implies that Ba2+ may bind at the intracellular channel surface.