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Glutamic acid decarboxylase (GAD), the synthesizing enzyme for the neurotransmitter GABA, has been localized in goldfish retina using a new antiserum. We observed at least six types of GAD-immunoreactive amacrine cells, one of which was large and pyriform (Ab type). In addition, immunoreactive synaptic terminals were located throughout the inner plexiform layer (IPL). Amacrine cells that were GAD-immunoreactive also had high-affinity uptake mechanisms for both 3H-GABA and 3H-muscimol that were detectable autoradiographically. Type Ab pyriform amacrine cells were heavily labeled because of 3H-GABA uptake and were GAD-immunoreactive. Other types of GAD-immunoreactive amacrine cells, including a subpopulation of Ab amacrines, were lightly labeled because of 3H-GABA uptake. Because the same neurons that were GAD-immunoreactive also accumulated 3H-GABA and 3H-muscimol, these three are appropriate markers for GABAergic cells in the goldfish retina. However, the uptake of 3H-muscimol by many non-GAD-immunoreactive cells, detectable at longer autoradiographic exposure times, indicates that this label must be used with caution. Thirty percent of goldfish retinal amacrine cells are GABAergic, and their processes are distributed throughout all levels of the IPL. Few GAD-immunoreactive amacrine cells accumulated 3H-glycine, so the goldfish retina contains distinct populations of glycinergic and GABAergic amacrine cells.