1. Single-channel currents activated by the glutamate agonist N-methyl-D-aspartate (NMDA) were recorded from outside-out patches of cultured rat cortical neurons in the presence of intracellular Mg2+ (Mgi2+). The rate constants of the block by Mgi2+ were measured using amplitude distribution analysis. 2. At a membrane potential of 0 mV, the blocking rate constant (k+) of Mgi2+ was estimated to be 2.1 x 10(7) M-1 S-1 and the unblocking rate constant (k-) 1.7 x 15(5)s-1. The very fast rate constants of the block by Mgi2+ explain why channel flicker was not fully resolvable during block of the NMDA-activated single-channel current by Mgi2+. 3. The blocking rate constant of Mgi2+ increased with increasing concentrations of Mgi2+. The unblocking rate constant was Mgi2+ concentration independent. 4. The blocking rate constant increased e-fold per 64 mV depolarization, whereas the unblocking rate constant decreased e-fold per 133 mV depolarization. The dissociation constant (KD) calculated from the blocking rates (k-/k+) decreased e-fold per 43 mV depolarization, and had a value at 0 mV of 7.8 mM. These values are consistent with previous estimates obtained from the voltage-dependent inhibition of the single-channel current amplitude. Both results predict, based on the Woodhull model, that Mgi2+ traverses about one-third of the membrane field to reach its blocking site. 5. The unblocking rate constant of Mgi2+ is one to two orders of magnitude faster than the previously reported unblocking rate constant of extracellular Mg2+ (Mgo2+) in the physiological voltage range, and their voltage dependencies are of opposite signs. These findings are consistent with the hypothesis that there are separate binding sites in the channel for Mgi2+ and Mgo2+. 6. Based on the blocking kinetics of Mgi2+ and Mgo2+, an energy profile of three barriers and two binding sites for Mg2+ is proposed.