The aim of this study was to determine from macroscopic current analysis how intracellular magnesium ions, Mgi2+, interfere with sodium channels of mammalian neurones. It is reported here that permeation across the sodium channel is voltage- and concentration-dependently reduced by Mgi2+. This results in a general reduction of sodium membrane conductance and an outward sodium peak current at large positive potentials. 30 mM Mgi2+ leads ot a negative shift of voltage dependence of sodium channel gating parameters, probably due to the surface potential change of the membrane. This shift alone is, however, insufficient to explain the reduction of outward sodium currents. The blockage by Mgi2+ is decreased upon increasing intracellular or extracellular Na+ concentration, which suggests that Mgi2+ interferes with sodium permeation by competitively occupying sodium channels. Using a kinetic model to describe the sodium permeation, the dissociation constant (at zero membrane potential) of Mgi2+ for the sodium channel has been calculated to be 8.65 +/- 1.51 mM, with its binding site located at 0.26 +/- 0.05 electrical distance from the inner membrane. This dissociation constant is smaller than that of Nai+, which is 83.76 +/- 7.60 mM with its binding site located at 0.75 +/- 0.23. The low dissociation constant of Mgi2+ reflects its high affinity for the sodium channel.