In the brain, the γ-aminobutyric acid (GABA) level is primarily controlled by the activity of its synthesizing enzyme, L-glutamate decarboxylase (GAD). At present, mechanisms responsible for regulation of GAD activity remain largely unknown. Here we report that GAD activity is inhibited by conditions favoring protein phosphorylation, and this inhibition can be reversed by phosphatase treatment. Furthermore, this inhibition appears to result from the suppression of a Ca^2+ -dependent phosphatase. Phosphorylation of GAD is demonstrated by direct incorporation of 32P into the GAD protein. These results suggest that GAD activity in the brain is inhibited by phosphorylation and activated by dephosphorylation. A model for regulation of GABA synthesis related to neuronal excitation is discussed.