It has been previously demonstrated that the majority of the glutamatergic input to directionally selective (DS) ganglion cells in the rabbit retina is mediated by NMDA receptors. To examine whether NMDA channels have any role in directional selectivity, we eliminated magnesium from the superfusion medium to prevent the magnesium block of the channels at hyperpolarized membrane potentials. During superfusion in magnesium-free media, the response to null-direction motion increased to the level of the response to preferred-direction motion. This effect was specifically mediated by NMDA channels because subsequent blocking of the NMDA channels with AP7 restored directional selectivity. We also tested whether the increase in the null-direction response in magnesium-free medium was due to an increased release of acetylcholine from the cholinergic amacrine cells, rather than an effect on the DS ganglion cells themselves, by blocking acetylcholine transmission with d-tubocurarine during superfusion with the magnesium-free medium. During zero-magnesium superfusion, d-tubocurarine reduced both the preferred- and null-direction responses of DS ganglion cells but did not restore directional selectivity. These findings suggest that null-direction motion normally causes portions of the dendritic membrane of the directionally selective ganglion cell to be maintained at a sufficiently negative potential that the NMDA channels are blocked by magnesium ions. This result is discussed in terms of several models for the mechanisms of directional selectivity.