Zinc ions (Zn(2+)) are localized in presynaptic vesicles at glutamatergic synapses and released in an activity-dependent manner. Modulation of NMDA-type glutamate receptors by extracellular Zn(2+) may play an important role under physiological conditions and during pathologies such as ischaemia or seizure. Zn(2+) inhibits NMDA receptors containing the NR2A subunit with an IC(50) value in the low nanomolar concentration range. Here we investigate at the single-channel level the mechanism of high affinity Zn(2+) inhibition of recombinant NR1/NR2A receptors expressed in HEK293 cells. Zn(2+) reversibly decreases the mean single-channel open duration and channel open probability determined in excised outside-out patches, but has no effect on single-channel current amplitude. A parallel series of experiments demonstrates that lowering extracellular pH (increasing proton concentration) has a similar effect on NR1/NR2A single-channel properties as Zn(2+). Fitting the sequence of single-channel events with kinetic models suggests that the association of Zn(2+) with its binding site enhances proton binding. Modelling further suggests that protonated channels are capable of opening but with a lower open probability than unprotonated channels. These data and analyses are consistent with Zn(2+)-mediated inhibition of NMDA receptors primarily reflecting enhancement of proton inhibition.