Intravenous administration of lipopolysaccharide (LPS) to rats increased the production of nitric oxide (NO) metabolites (NO_x) by blood polymorphonuclear neutrophils (PMN) in vitro. Both dexamethasone and L‐NMMA, added in vitro to neutrophil cultures, inhibited the production of NO. On the other hand, the production of NO was not affected by the treatment, in vivo or in vitro, with different inhibitors of cyclooxygenase or 5‐lipoxygenase or with a platelet‐activating factor (PAF) antagonist. The incubation of blood PMN from normal rats in vitro with neutrophil activators (PAF, leukotriene B₄, and interleukin‐8) and different cytokines [interleukin‐1, tumor necrosis factor α, and interferon‐γ (IFN‐γ)] showed that only IFN‐γ was able to induce the production of high amounts of NO. This induction was directly correlated with the expression of iNOS and an increase in in the enzyme activity in blood PMN. The tyrosine kinase inhibitor genistein inhibited NO production induced by IFN‐γ, suggesting that the signal transduction pathway leading to NOS induction in rat PMN involves phosphorylation by tyrosine kinase. We also showed that NO produced by IFN‐γ activated rat blood PMN involved in the killing of Pseudomonas aeruginosa. J. Leukoc. Biol. 65: 508–514; 1999.