Immunostaining and high-pressure liquid chromatography (HPLC) were used to study the developmental time course of astrocytic gamma-aminobutyric acid (GABA) expression in rat optic nerve. GABA immunostaining was carried out on cultured astrocytes, and on whole optic nerve. Confocal scanning laser microscopy was used to obtain optical sections in excised whole tissue in order to localize the cellular origins of GABA within the relatively intact optic nerve. GABA immunoreactivity was localized in astrocytes identified by GFAP staining; GABA staining was most intense in early neonatal optic nerve and attenuated over 3 weeks of postnatal development. The staining was pronounced in the astrocyte cell bodies and processes but not in the nucleus. There was a paucity of GABA immunoreactivity by postnatal day 20, both in culture and in whole optic nerve. A biochemical assay for optic nerve GABA using HPLC indicated a relatively high concentration of GABA in the neonate, which rapidly attenuated over the first 3 postnatal weeks. Immunoreactivity for the GABA synthesis enzyme glutamic acid decarboxylase (GAD) was pronounced in neonates but also attenuated with development. These results indicate that GABA and the GABA synthesis enzyme GAD are localized in astrocytes of optic nerve, and that their expression is transient during postnatal development.