Abstract : Astrocytes play a pivotal role in CNS detoxification pathways, where glutathione (GSH) is involved in the elimination of oxygen and nitrogen reactive species such as nitric oxide. We have previously demonstrated that the specific activity of γ-glutamyl transpeptidase (γ-GT), an enzyme of central significance in GSH metabolism, is regulated in vivo in astrocytes by 1,25-dihydroxyvitamin D3 (1,25-D3). The aim of the present work was to investigate, in primary cultures of newborn rat astrocytes, the effects of this hormone on γ-GT synthesis and on GSH and nitrite levels after lipopolysaccharide (LPS) treatment. This study demonstrates that both γ-GT gene expression and specific activity, induced by LPS, are potentiated by 1,25-D3. In contrast, 1,25-D3 does not regulate the expression of other enzymes involved in astrocyte detoxification processes, such as superoxide dismutase or GSH peroxidase. In parallel, 1,25-D3 enhanced intracellular GSH pools and significantly reduced nitrite production induced by LPS. Taken together, these results suggest that γ-GT, GSH, and 1,25-D3 play a fundamental role in astrocyte detoxification pathways.