To clarify the properties of an inducible type of nitric oxide synthase (i-NOS) in the brain, we examined whether lipopolysaccharide (LPS) induces NOS in glial cells cultured from neonatal rats. NOS activities (NO2- accumulation and L-[14C]citrulline formation) were detected by treatment with LPS at 10 micrograms/ml for 6-72 hr. L-[14C]citrulline formation by LPS-induced i-NOS was inhibited by NG-monomethyl-L-arginine (a NOS inhibitor) and diphenyleneiodonium (a flavo-protein inhibitor). The activity was not markedly changed in the presence or absence of Ca2+. The induction of i-NOS by LPS was abolished by cycloheximide, actinomycin D, or dexamethasone. In addition, the induction was inhibited by herbimycin A (a tyrosine kinase inhibitor), but was not by staurosporine, W-7, or FK-506. After LPS stimulation, 130 kDa proteins were reacted with anti-rat liver i-NOS antibody 5-72 hr. i-NOS induced from glial cells coupled tightly with endogenous calmodulin (CaM) even in the absence of Ca2+. These results suggest that LPS induces expression of 130-kDa i-NOS through an activation of tyrosine kinase, after which i-NOS couples with CaM, and that NO is formed for 6-72 hr in glial cells.