1. The molecular determinants for the narrow constriction of recombinant N-methyl-D-aspartate (NMDA) receptor channels composed of wild-type and mutant NR1- and NR2A-subunits were studied in Xenopus oocytes. 2. The relative permeability of differently sized organic cations was used as an indicator of the size of the narrow constriction. From measured reversal potentials under bi-ionic conditions with K+ as the reference solution, permeability ratios were calculated with the Lewis equation. 3. For wild-type NMDA receptor channels, five organic cations showed clear reversal potentials, with permeability ratios (PX/PK): ammonium, 1.28; methylammonium, 0.48; dimethylammonium (DMA), 0.20; diethylammonium, 0.07; and dimethylethanol-ammonium, 0.02. 4. Mutation of the N-site asparagine (N) to glutamine (Q) at homologous positions in either NR1 (position 598) or NR2A (position 595) increased the permeability of DMA relative to wild-type channels about equally. However, for larger sized organic cations, the NR1(N598Q) mutation had stronger effects on increasing their permeability whereas the NR2A(N595Q) mutation was without effect. These changes in organic cation permeability suggest that the NR1(N598Q) mutation increases the pore size while the NR2A(N595Q) mutation does not. 5. Channels in which the NR1 N-site asparagine was replaced by the smaller glycine (G), NR1(N598G)-NR2A, showed the largest increase in pore size of all sites examined in either subunit. In contrast, in the NR2A-subunit the same N-site substitution to glycine produced only small effects on pore size. 6. For the NR2A-subunit, an asparagine residue (position 596) on the C-terminal side of the N-site, when mutated to larger or smaller sized amino acids, produced large, volume-specific effects on pore size. The mutant channel NR1-NR2A(N596G) had the largest increase in pore size of all sites examined in the NR2A-subunit. In contrast, mutation of the homologous position in the NR1-subunit had no effect on pore size. 7. The cross-sectional diameter of the narrow constriction in wild-type NMDA receptor channels was estimated to be 0.55 nm. The pore sizes of the NR1(N598G)-NR2A and NR1-NR2A(N596G) mutant channels increased to approximately 0.75 and 0.67 nm, respectively. The double mutation, NR1(N598G)-NR2A(596G), increased the pore size to approximately 0.87 nm, essentially the sum of the increase produced by the individual mutations. 8. It is concluded that both the NR1- and NR2A-subunits contribute to the narrow constriction of NMDA receptor channels with asparagines located at non-homologous positions. The major determinants of the narrow constriction in NMDA receptor channels are the NR1 N-site asparagine and an asparagine adjacent to the NR2A N-site.