Glutamic acid decarboxylase (GAD), the gamma-aminobutyric acid (GABA)-synthetic enzyme, consists of two isoforms, GAD67 and GAD65. Although distributions of the two GAD isoforms at the somatic level are known to be heterogeneous among different subpopulations of GABAergic neurons, those at the synaptic level have not been investigated. In order to analyze quantitatively the two GAD-isoform immunoreactivities in axon terminals, we combined confocal laser scanning microscopy with digitized image analysis to measure the gray levels of immunofluorescent signals for the two GAD isoforms in a large number of individual boutons in each hippocampal and dentate layer of the mouse. Synaptic boutons exhibited lamina-specific immunoreactivities against the GAD isoforms. Boutons in the principal cell layers (stratum pyramidale of the hippocampus proper and the granule cell layer of the dentate gyrus) showed more intense immunoreactivity against GAD67 than those in the dendritic layers (strata lacunosum-moleculare, radiatum, and oriens of the hippocampus proper and the molecular layer of the dentate gyrus). By contrast, boutons in the dendritic layers showed more intense immunoreactivity against GAD65 than those in the principal cell layers. Such differential distributions could be correlated to the GAD-isoform immunoreactivities in the axon terminals originating from parvalbumin-containing neurons, a particular subpopulation of hippocampal GABAergic neurons mainly innervating the perisomatic domain of principal neurons. In addition to previously reported physiological and pharmacological differences between the GABAergic synapses on perisomatic domain and those on distal dendrites, the present results suggest a functional differentiation of GABAergic synapses between these two inhibitory sites.