Although glutamate is considered the natural neurotransmitter that mediates excitatory function in the CNS, other active natural compounds can also drive the functional activation of excitatory amino acid receptors (EAAR). L-aspartate is the most likely neurotransmitter to mimic the actions of glutamate. Here we show that L-aspartate promotes the release of GABA acting selectively on the NMDA receptor subtype. Retina cell cultures, when exposed to excitatory amino acids (EAA), release [3H] GABA previously incorporated by the cells. Both L-glutamate and L- and D-aspartate at 100 microM concentration, promote the release which can be mimicked by kainate and NMDA. While aspartate-induced release of [3H] GABA occurs in the presence of 1 mM Mg2+, NMDA (100 microM) promotes the release only when Mg2+ is omitted from the superfusing medium. However, in the absence of Mg2+ the efficacy of 1- and d-aspartate (100 microM) to activate [3H] GABA release increases by a factor of 2 when compared to the release observed in the presence of 1 mM Mg2+. NMDA and aspartate induced release of [3H] GABA is completely inhibited by 10 microM MK-801 and is not affected by CNQX (100 microM). In the presence of Mg2+, aspartate-induced release of [3H] GABA is also completely inhibited by MK-801 (10 microM) and is not significantly affected by CNQX (100 microM). The [3H] GABA release induced by kainate (100 microM) is fully inhibited by CNQX (100 microM) and is not affected by MK-801 (10 microM). Our results indicate that in the retina, l-aspartate modulates its excitatory function on a set of GABAergic cells via the selective activation of NMDA receptors. The fact that L- and D-aspartate (but not D-glutamate) induce the release of GABA even in the presence of Mg2+ suggests that the electrogenic uptake of aspartate is required to lower the affinity of the NMDA channel for Mg2+. The observation that D-glutamate (200 microM), which is not taken up by the cells, activates the efflux of GABA only when Mg2+ is omitted from the incubating medium, supports this possibility.