The emergence of GABA-accumulating neurons was studied from stages 29 to 40 during retinal histogenesis in the chick, covering embryonic (E) days E6-E14, using autoradiographic analysis following incubation of isolated retinas with [3H]GABA (2 microM). Analysis was restricted to central retina which is more advanced in its differentiation than the periphery. On E6 numerous mitotic figures were present along the scleral border of the unstratified neuroepithelium. Specific localization of [3H]GABA was associated initially with somata situated in middle regions of the retinal expanse. Occasionally contiguous pairs of labeled cells were seen. The inner plexiform layer makes its appearance during E7; at that time silver grains were present over cell bodies located in the ganglion cell layer and the proximal portion of the inner nuclear layer, those of probable amacrine cells. As retinal stratification continued, more cells were observed to have elaborated membrane systems for GABA uptake with varying degrees of affinity. By E8, although dividing, non-labeled cells were in close proximity, GABA-labeled cells were observed in positions of horizontal cells. By E14, the pattern of label distribution appeared essentially similar to that reported for adult retina, i.e. [3H]GABA labeling was observed over horizontal cells and their processes, subpopulations of amacrine cells which appear to ramify extensively across the inner plexiform layer, selected perikarya of the ganglion cell layer, and the nerve fiber layer. In addition, a subpopulation of labeled photoreceptors, some identified as cones by virtue of oil droplets, was observed. Thus, preferential accumulation of GABA appears during E6, prior to formation of either inner or outer plexiform layers. The localization of [3H]GABA demonstrates that ganglion and amacrine cell bodies are labeled initially, followed by horizontal cells. Specific accumulation of [3H]muscimol, a potent agonist of GABA receptors, appears about E12 over cells located in proximal regions of the inner nuclear layer; these somata later ramify in sublaminae 2 and 4 of the inner plexiform layer.