The putative neurotransmitter gamma-aminobutyric acid (GABA) was localized in goldfish retina by using an antiserum directed against GABA itself. The same types of cells were stained with this antibody as were labelled with an antiserum directed against the synthesizing enzyme for GABA, glutamic acid decarboxylase. Stained neurites of these cells were located throughout the inner plexiform layer (IPL) but staining was more intense in the proximal IPL. The GABA-immunoreactive staining could be reduced or completely abolished by preabsorbing the primary antibody with GABA. Uptake of [3H]-GABA or the GABA agonist [3H]-muscimol was localized in GABA-stained retinas using light microscope autoradiography. These experiments demonstrated that all types of GABA-immunoreactive amacrine cells had high-affinity uptake mechanisms for both [3H]-GABA and -muscimol. Thirty percent of proximal inner nuclear layer (INL) and some cells in the ganglion cell layer (GCL) were labelled by all three GABAergic markers. Most GABA-immunoreactive amacrine cells were lightly labelled due to [3H]-GABA uptake but a few amacrines (Ab) were heavily labelled. These findings demonstrate that the autoradiographic localization of [3H]-GABA or [3H]-muscimol uptake and the immunocytochemical localization of GAD or GABA are appropriate methods for localizing GABAergic neurons in the retina. Few GABA-immunoreactive amacrine cells accumulated the putative amino acid transmitter [3H]-glycine, verifying that the goldfish retina contains distinct subpopulations of glycinergic and GABAergic amacrine cells.