We studied the effect of Ca 2 + on the transport of the γ-aminobutyric acid (GABA) by synaptic plasma membrane (SPM) vesicles isolated from sheep brain cortex and observed that intravesicular Ca 2 + inhibits the [ 3 H]GABA accumulation in a concentration-dependent manner. This inhibitory effect of Ca 2 + exhibited two distinct components: one in the micromolar range of Ca 2 + concentration, and the other in the millimolar range. Previous EGTA washing of the membranes, or incorporation of trifluoperazine into the vesicular space reduced the inhibitory action of Ca 2 + , particularly at low Ca 2 + (1-5 μM). Okadaic acid (1 μM) also relieved the Ca 2 + inhibition at low, but not at high Ca 2 + concentrations (1 mM), whereas the calpain inhibitor I did not alter the effect of the low Ca 2 + , but it partially reduced ( 28%) the effect of Ca 2 + in the millimolar range. The results indicate that the GABA transporter is regulated by low Ca 2 + concentration (μM) and probably its effect is mediated by the (Ca 2 + calmodulin)-stimulated phosphatase 2B (calcineurin). In contrast, the GABA uptake inhibition observed at high Ca 2 + concentrations (1 mM) is less specific, and probably it is partially related to the proteolytic activity of membrane bound calpain II.