In GAD65-knockout mice, lack of GAD65 expression was confirmed. The expression level of vesicular GABA transporter (VGAT) was upregulated, and no change in the synaptic vesicles (SV)-associated GAD67 was found. GAD65(-/-) SV transported cytosolic GABA much more efficiently than that of the wild type, further supporting our model that there is a structural and functional coupling between GABA synthesis and packaging into SV. Both full-length and truncated forms of GAD65 could bind to GABAergic SV, indicating the N-terminus is not required for the anchoring of GAD65 to SV. Although both GAD65(-/-) SV reconstituted with either GAD65 or GAD67 could synthesize GABA from [3H] glutamate and transport this newly synthesized GABA into SV, the combined evidence suggests that GAD65 plays a major role in GABA transmission in normal physiological condition. However, GAD67 could serve this role under some pathological conditions.