GAD65, the smaller isoform of the γ-aminobutyric acid-synthesizing enzyme glutamic acid decarboxylase is detected as an α/β doublet of distinct mobility on SDS-polyacrylamide gel electrophoresis. Glutamic acid decarboxylase (GAD) 65 is reversibly anchored to the membrane of synaptic vesicles in neurons and synaptic-like microvesicles in pancreatic β-cells. Here we demonstrate that GAD65α but not β is phosphorylated in vivo and in vitro in several cell types. Phosphorylation is not the cause of the α/β heterogeneity but represents a unique post-translational modification of GAD65α. Two-dimensional protein analyses identified five phosphorylated species of three different charges, which are likely to represent mono-, di-, and triphosphorylated GAD65α in different combinations of phosphorylated serines. Phosphorylation of GAD65α was located at serine residues 3, 6, 10, and 13, shown to be mediated by a membrane bound kinase, and distinguish the membrane anchored, and soluble forms of the enzyme. Phosphorylation status does not affect membrane anchoring of GAD65, nor its Km or Vmax for glutamate. The results are consistent with a model in which GAD65α and -β constitute the two subunits of the native GAD65 dimer, only one of which, α, undergoes phosphorylation following membrane anchoring, perhaps to regulate specific aspects of GAD65 function in the synaptic vesicle membrane.