The influx and exchange of glycine were studied in synaptic plasma membrane vesicles isolated from rat brain. The vesicles were loaded with [U-14C]glycine by active transport driven by an Na+ as well as a Cl- gradient (out greater than in). Dilution-induced efflux requires the simultaneous presence of internal Na+ and Cl-. As the efflux of glycine has been demonstrated to be strictly dependent on the presence of both ions (Mayor, F., Jr., Marvizón, J.G., Aragón, M.C., Giménez, C. and Valdivieso, F. (1981) Biochem. J. 198, 535-541), it can be concluded that the efflux of glycine is, in many aspects, symmetrical with its influx. Glycine efflux from the membrane vesicles is stimulated by external glycine, this exchange being partially dependent on external sodium and not on external chloride. The parallelism observed in influx and efflux processes shows that the release of glycine (by efflux and homoexchange) occurs via the carrier system. These results suggest that glycine is translocated in both directions across the membrane, probably by interacting with the carrier. Both sodium and chloride have to be present on the same side as glycine.