The distribution of the neuroactive amino acids glutamate, GABA, and glycine in the human retina was examined in consecutive semithin sections treated with antisera specific for fixed glutamate, GABA, and glycine, respectively. Glutamate immunoreactivity was conspicuous in all photoreceptor cells (rods more strongly labelled than cones), and in a majority (85-89%) of the cells in the inner nuclear layer (INL). Rod spherules and cone pedicles showed a greater enrichment of glutamate immunoreactivity than the parent cell bodies and inner segments. Also, structures of the inner plexiform layer (IPL) were labelled. A large majority (83-91%) of cells in the ganglion cell layer (GCL) was strongly stained, as were most axons in the nerve fibre layer. Müller cell processes appeared unstained. GABA immunoreactivity was present in presumed amacrine but not in bipolar-like cells. The stained cells were restricted to the inner 1/3 of the INL and were more frequent in central than in peripheral retina (40% and 26% of all cells in the inner 1/2 of INL, respectively). GABA positive cell processes, probably originating from interplexiform cells, appeared to traverse the INL and end in the outer plexiform layer. Dense immunolabeling was found in the IPL. GABA immunoreactive cells (some also stained for glutamate) comprised 23% of all GCL cells in the peripheral retina, but only 5% in the central retina. Most of them were localized adjacent to the IPL. A few GABA positive (possibly ganglion) cells extended a single fibre toward the nerve fibre layer. Solitary GABA positive fibres were seen in this layer and in the optic nerve. Glycine immunoreactivity was observed in cells with the location typical of amacrine and bipolar (peripheral retina) cells, as well as in punctate structures of the IPL. In contrast to the GABA positive cells, the glycine positive cells were more frequent in the peripheral than in the central retina (42% and 23% of all cells in inner 1/3 of INL, respectively). A few cells in the GCL (0.5-1.5%) were glycine positive. Glutamate colocalized with GABA or glycine in a majority of the cells stained for either of these inhibitory transmitters (90-95% of the GABA positive cells, and 80-86% of the glycine positive cells, in the INL). Some bipolar cells were stained for both glutamate and glycine. Colocalization of GABA and glycine occurred in a subpopulation (3-4%) of presumed amacrine cells, about half of which was also glutamate positive.