Isolated horizontal cells from goldfish retinas were prepared by enzymatic dissociation using papain and separated from other cells by velocity sedimentation. In the intact retina, H1 horizontal cells possess a high-affinity mechanism for accumulating gamma-aminobutyric acid (GABA). This property is retained in isolated cells, which also release the accumulated GABA in response to depolarization by elevated external K+. L-Glutamic acid and its analogues are highly effective at micromolar concentrations in eliciting the release of preloaded GABA from isolated cells. At saturating concentrations, L-aspartic acid stimulates about one-third as much release as L-glutamic acid. In contrast, the D-isomers of glutamate and aspartate are ineffective. In the intact retina, micromolar concentrations of L-glutamic acid analogues are also capable of eliciting GABA release from H1 horizontal cells. Release of the accumulated GABA from isolated H1 cells is largely independent of external Ca2+ concentrations. In the intact retina, H1 horizontal cells also possess a K+-stimulated GABA release mechanism that is independent of the Ca2+ concentrations in the medium. In addition, there appears to be a small but significant amount of [3H]GABA release that may be Ca2+ dependent. Under our conditions, [3H]GABA release from isolated cells is unaffected by external Na+ concentrations between 20 and 120 mM. However, concentrations of 10 mM or less significantly diminishes this release, with 70% curtailed in Na+-free solutions. Our results, together with morphological observations by a number of other investigators, suggest that there may be two distinct mechanisms for GABA release from goldfish H1 horizontal cells: one being a conventional vesicular mechanism which is Ca2+ dependent, while the other is Na+ driven and Ca2+ independent. H1 horizontal cells in the intact goldfish retina release the accumulated GABA in response to brief incubations in darkness, which is known to be the natural stimulus that depolarizes these neurones.