Abstract Electrical stimulation of rat spinal cord slices superfused in vitro produced a greater than two-fold increase in the rate of efflux of glycine and GABA. No urea was released by electrical stimulation. There was a small increase in the rate of efflux of proline, llysine, and cycloleucine, a somewhat greater increase in the rate of efflux of glutamic acid, but the only other amino acid that compared to glycine and GABA in extent of release was taurine. No regional specificity of electrically induced release was found for glycine or GABA, whereas there was a significantly greater release of glutamic acid from cerebral cortex than from spinal cord or cerebellum. High potassium (30–60 m M) or low sodium (28 m M) in the superfusion medium did not influence the electrically stimulated release of glycine or GABA from spinal cord slices. Incubation of slices in standard medium followed by superfusion with Ca-free medium had no effect on stimulated release of labeled glycine or GABA. However, when the slices were incubated and superfused in Ca-free medium containing 2 m M EDTA there was a marked reduction in electricity stimulated release of glycine and GABA. Superfusion with 1 m M strychnine had no effect. Cyanide reduced the release of glycine but not of GABA. Ouabain in the superfusion medium Completely abolished the release of glycine and GABA with electrical stimulation.