In the mammalian retina, high levels of the group II metabotropic glutamate receptor (mGluR) subtype are expressed in starburst amacrine cells. A prominent role of starburst amacrine cells is the generation of directional selectivity in ON-OFF directionally selective retinal ganglion cells (DS RGCs). Extracellular microelectrodes were used to study the effects of activation of group II mGluRs on the responses of rabbit ON-OFF DS RGCs to a moving light stimulus. Directionally selective responses in these RGCs were substantially reduced by the selective group II mGluR agonist DCG-IV. DCG-IV brought out a response to movement in the null direction that was similar in magnitude and time course to the response to movement in the preferred direction. This effect of DCG-IV was reversed by the group II mGluR antagonist EGLU. Application of EGLU alone failed to alter directional selectivity in the RGCs but did reduce the response to movement in the preferred direction. To determine whether group II mGluRs modulate the release of the neurotransmitter acetylcholine from starburst amacrine cells, the effect of DCG-IV on ON-OFF DS RGCs was examined in the presence of ambenonium, an acetylcholinesterase inhibitor. When applied alone, ambenonium greatly prolonged the responses of ON-OFF DS RGCs to a light stimulus. This effect of ambenonium was completely abolished upon application of DCG-IV. Overall, the results suggest that postsynaptic group II mGluRs have the potential to influence directional selectivity in RGCs by inhibiting transmitter release from starburst amacrine cells.