The Ca(2+) permeability of N-methyl-D-aspartate receptor (NMDA-R) channels was studied in human embryonic kidney cells transfected with the NR1-NR2A subunit combination. To determine the fractional Ca(2+) current (P(f)), measurements of fura-2-based Ca(2+) influx and whole-cell currents were made in symmetrical monovalent ion concentrations at membrane potentials between -50 mV and the reversal potential. The ratios of Ca(2+) flux over net whole-cell charge at 2, 5, and 10 mM external Ca(2+) concentrations ([Ca](o)) were identical at a membrane potential close to the reversal potential of the monovalent current component. Assuming unity of P(f) at this potential, the percentage of current carried by Ca(2+) was found to be 18.5 +/- 1.3% at 2 mM [Ca](o) and -50 mV. This value, which is higher than the ones reported previously, was confirmed in independent experiments in which a pure flux of Ca(2+) through NMDA-R channels was used to calibrate the Ca(2+) influx signals. The measured values of fractional Ca(2+) currents, which agree with the predictions of the Goldman-Hodgkin-Katz equations, are also compatible with a two-barrier model for ion permeation, in which the differences between the energy barriers for Ca(2+) and monovalent ions are similar on the external and internal membrane sides.