Cytokines are released in the central nervous system following brain injury and disease. Several of those conditions are thought to involve the accumulation of extracellular glutamate at excitotoxic concentrations, and may involve compromised glial glutamate uptake. Using primary cultures of postnatal rat hippocampus, we studied the effect of three cytokines on astrocytic high-affinity glutamate uptake. After 24 hours incubation with either tumor necrosis factors alpha (TNF-alpha), interferon gamma (IFN-gamma) or interleukin-1 beta (IL-1 beta), astrocytic glutamate uptake was markedly attenuated in a dose-dependent manner. Cytokine effects were reversed by inhibition of nitric oxide synthase (NOS) using N omega-nitro-L-arginine (LNA), NG-monomethyl-L-arginine acetate (L-NMMA) or N omega-nitro-L-arginine methyl ester (LNAME). Moreover, application of the NO donors 3-morpholinosydnonimine (SIN-1) and s-nitroso-n-acetylpenicillamine (SNAP) mimicked cytokine inhibition of glutamate uptake. These data suggest that cytokine release can inhibit astrocytic glutamate uptake through a pathway that involves the liberation of nitric oxide. Astrocytic glutamate uptake may thus be compromised under conditions that are known to cause cytokine release such as nervous system injury, inflammation and ischemia.