Appearance and maturation of the GABA (gamma-aminobutyric acid) system during newt retinal regeneration were studied by electrophysiological, immunohistochemical, and biochemical techniques. (1) Responses to GABA appeared in neurons dissociated from regenerating retinae before the segregation of the plexiform layers; whereas (2) GABA immunoreactivity appeared at sites of the presumptive horizontal cell and amacrine cell layers at the beginning of the segregation of these layers. During subsequent regeneration, GABA-immunoreactive cells at the amacrine cell layer increased in number and extended lateral processes, forming a GABA-immunoreactive inner plexiform layer. Also GABA immunoreactivity increased in the region of the outer plexiform layer, but not their somata which showed decreased GABA immunoreactivity. (3) GABA synthesis in the retina increased significantly at the beginning of the segregation of the plexiform layers. These results suggest that the increase of GABA synthesis during retinal regeneration correlates well with the development of GABA-immunoreactive cells and that functional GABA receptors appear earlier than increased GABA synthesis.