In primary cultures of rat astroglial cells exposure to bacterial endotoxin lipopolysaccharide (LPS) causes induction of a Ca(2+)-independent form of the nitric oxide synthase (iNOS) enzyme. We have now cloned the mRNA encoding astroglial iNOS using a combination of cDNA library screening and polymerase chain reaction (PCR) amplification with degenerate oligonucleotides directed against conserved regions of all NOS enzymes. The sequence of astroglial iNOS cDNA is highly similar to the mouse macrophage sequence, having an overall homology of 92% at the DNA level and 93% at the protein level. As in other NOSs, canonical binding sites for redox cofactors are present. The 3'-untranslated region displays 4 consensus AU-pentamers, 2 polyadenylation sites, and terminates in a stretch of 17 adenosine residues. In situ hybridization studies with LPS-treated astrocyte cultures demonstrated the presence of iNOS mRNA in the majority of astroglial cells, identified by antibody staining to the glial fibrillary acidic protein (GFAP). PCR analysis showed that LPS stimulated synthesis of astrocyte iNOS mRNA, which was detected as early as 2 hr after exposure to LPS, peaked at 4 hr, and slowly declined over the next 20 hr. These results confirm that astrocytes can express iNOS and provide tools for the subsequent analysis of iNOS gene expression in rodent brain.