A total of 5-6 different cell types in vertebrate retinas accumulate [3H]gamma-aminobutyric acid (GABA). In frog retina, specific populations of cells in the horizontal, amacrine and ganglion cell layers are labeled autoradiographically after a 15-min in vitro incubation with [3H]GABA. Cells which may be bipolar or interplexiform cells are also labeled. Similar autoradiographic patterns are observed in chick retina except for the absence of labeled bipolar or interplexiform cells. In rat retinas, [3H]GABA uptake is limited primarily to Muller and amacrine cells. Depolarizing glutamate receptor agonists (glutamate, aspartate and kainic acid) applied in an in vitro perfusion system, stimulated massive release of [3H]GABA from frog and chick retina but not from rat retina. Under these conditions, autoradiographic labeling of horizontal cells was virtually depleted, while labeling of other cell types remained robust. In contrast, potassium caused release of the label from all 3 types of retina, and loss of autoradiographic labeling occurred uniformly in all cell types. We conclude that [3H]GABA-accumulating horizontal cells possess depolarizing glutamate receptors and that activation of these receptors leads to a release of GABA stores. On the other hand, Muller cells and the various subclasses of [3H]GABA-accumulating amacrine, bipolar and/or interplexiform cells, do not release GABA in response to glutamate receptor stimulation and thus appear to be relatively insensitive to excitatory amino acids.