In the present study, we found that S-nitroso-N-acetyl-DL-penicillamine, a spontaneous nitric oxide (NO) generator, dose-dependently inhibited basal nerve growth factor (NGF) release from mixed glial cells. To elucidate the function of endogenous NO in the regulation of NGF release, the mixed glial cells were stimulated with lipopolysaccharide (LPS) or LPS plus interferon-gamma (IFNgamma). The results showed that LPS alone induced NGF release and moderate NO production. However, costimulation with LPS plus IFNgamma greatly enhanced NO production but significantly suppressed LPS-induced NGF release. When N(G)-monomethyl-L-arginine, an NOS inhibitor, was added to the culture, the suppression of NGF release by IFNgamma was significantly reduced. Quantitative reverse transcription-polymerase chain reaction demonstrated S-nitroso-N-acetyl-DL-penicillamine was also able to inhibit the LPS-induced NGF mRNA expression. To understand the different contributions of astroglia and microglia to this phenomenon, both cell types were purified. We found purified astroglia produced high amounts of NGF but low amounts of NO. However, purified microglia produced a large amount of NO but very low amounts of NGF after stimulation with LPS or LPS plus IFNgamma. Our data also indicated the second messenger cyclic GMP, but not cyclic AMP, was able to inhibit basal NGF release. In vivo experiments confirmed that NGF protein level was significantly enhanced in rats treated with L-N(omega)-nitro-arginine methyl ester and in endothelial NO synthase mutant mice. Taken together, we conclude NO derived mainly from microglia down-regulates NGF release from astroglia at the transcriptional level by stimulating cyclic GMP pathway.