The effect of ouabain on the uptake of tritiated [3H]GABA and on its release in the absence of Ca(+)+ was studied in brain cortex synaptosomes. Ouabain, in the absence of Ca(+)+ and K(+)-depolarization, induces the release of [3H]GABA with half-maximal effect occurring at a concentration of about 7 X 10(-6) M. Parallel measurements of the effects of ouabain on the [3H]GABA uptake and the Na+,K(+)-adenosine triphosphatase activity show that ouabain inhibits both mechanisms and that the half-maximal effect also occurs at about the same ouabain concentration. Although [3H]GABA release is stimulated by ouabain, it appears that the inhibition of [3H]GABA uptake is due to a direct effect on the uptake mechanism, inasmuch as the initial velocity of the process is inhibited by ouabain. Ouabain requires extracellular Na+ for [3H]GABA release and for membrane depolarization and, in the absence of Na+, ouabain does not cause either [3H]GABA release or membrane depolarization. No significant changes in the Na+ gradients occur under conditions which permit release of [3H]GABA, but the Na+,K(+)-adenosine triphosphatase activity is inhibited, which may be responsible for membrane depolarization, which in turn may cause [3H]GABA release or inhibit its uptake.