Previous studies in the rabbit retina have shown that drugs which block AMPA glutamate receptors abolish directional selectivity in ON–OFF directionally selective (DS) ganglion cells. The effects of activation of AMPA receptors on the directionally selective responses of these ganglion cells had not been studied. In the present study, extracellular recordings of the responses of ON–OFF DS ganglion cells to a moving bar of light were made in an in vitro rabbit retinal preparation. In control solution, bath application of AMPA (7–10 μM) abolished the light responses of most ON–OFF DS ganglion cells. On washout of AMPA, the light responses rapidly returned; however, the cells temporarily lost the ability to discriminate the direction of the moving bar of light. That is, the cells responded equally to movement in the preferred and null directions. Pretreatment of retinas with the glycine receptor antagonist strychnine (1–2 μM) did not alter the effects of AMPA. On the other hand, in retinas pretreated with the GABAA receptor antagonist SR95531 (0.2–0.25 μM), AMPA did not abolish the light responses of ON–OFF DS ganglion cells but instead abolished directional selectivity in these cells by bringing out a response to movement in the null direction. This finding suggests that an AMPA-induced GABA efflux from cells in the retina was responsible for the suppression of the light responses by AMPA. In control solution, application of the selective AMPA receptor agonist (S)-5-fluorowillardiine (2–3 μM) only temporarily abolished the light responses of ON–OFF DS ganglion cells. As the light responses returned, it was clear that directional selectivity had been abolished by (S)-5-fluorowillardiine. In control solution, blocking AMPA receptor desensitization with cyclothiazide (80–100 μM) greatly reduced the light responses of ON–OFF DS ganglion cells. As the light responses slowly returned on washout of cyclothiazide, directional selectivity was clearly reduced although not abolished. In retinas pretreated with SR95531, application of cyclothiazide abolished directional selectivity. Diazoxide (700–1000 μM), another blocker of AMPA receptor desensitization, abolished directional selectivity in ON–OFF DS ganglion cells without the need of adding SR95531 to the bathing solution. It is concluded that, in the rabbit retina, AMPA receptors play an important role in generating directional selectivity in ON–OFF DS ganglion cells. Moreover, excessive activation of AMPA receptors greatly compromises the mechanism for directional selectivity in ON–OFF DS ganglion cells.