We examined whether the cytokine IL-12 could be induced locally in the brain or in glial cell cultures following LPS treatment. In the brain, expression of IL-12 p35 mRNA was constitutive and did not alter following i.p. injection of LPS. In contrast, IL-12 p40 mRNA was only detectable in the brain of mice given two staggered injections of LPS. Dual labeling in situ analysis revealed IL-12 p40 RNA-positive cells scattered throughout the brain parenchyma, with a small number of these cells being identified as astrocytes, while the majority of IL-12 p40 RNA-expressing cells appeared to be microglia. In cultured microglia or astrocytes, LPS and to a much lesser degree IL-1beta, but not IFN-gamma or TNF-alpha, induced the expression of IL-12 p40 mRNA. Numerous glial fibrillary acidic protein-immunopositive cells colabeled for IL-12 p40 RNA; indicating that LPS-stimulated astrocytes expressed IL-12 in vitro. Immunoblot analysis of lysates from LPS-treated astrocytes revealed the presence of multiple species of 40, 43, 75, and 120 kDa containing the IL-12 p40 protein. Finally, secretion of the IL-12 p75 heterodimer was detectable by ELISA from astrocytes treated with LPS plus IFN-gamma, but not with LPS alone. The findings indicate that IL-12 gene expression can be activated in the brain, with the resident glial cells being a prodigious source of this cytokine. The localized production of IL-12 may have a significant impact on the development of cell-mediated immune responses within the central nervous system.