The Ca2+ permeability properties of an N-methyl-D-aspartate (NMDA) channel pore mutant (NR1E603K-NR2A) were studied using whole-cell patch-clamp recordings in human embryonic kidney cells. Measurements of reversal potential shifts indicated that the relative permeability of Ca2+ over monovalent ions, P(Ca)/P(M), was 1.6, a value reduced by a factor of approximately 2 with respect to the wild-type channel. The ratio of Ca2+ current over total current (fractional Ca2+ current), however, was 19.7 +/- 1% at -50 mV and 2 mM external Ca2+ concentration, a value similar to that of the wild-type channel, but 2.3-fold larger than that predicted by simple permeation models for the corresponding P(Ca)/P(M) value. The deviation from predicted values gradually disappeared with membrane depolarization. Similar results were obtained for two cysteine mutations at asparagine residues of the NR1 and NR2A subunits. When interpreted in terms of a two-barrier one-site model for ion permeation, the results indicate that changes in the relative Ca2+ permeability occur close to an internal energy barrier limiting ion permeation.