Nitric oxide (NO) produced by macrophages (Mφ) in response to interferon-γ (IFN-γ) plays a pivotal role in the control of intracellular pathogens. Current knowledge of the specific biochemical cascades involved in this IFN-γ-inducible Mφ function is still limited. In the present study, we evaluated the participation of various second messengers – Janus kinase 2 (JAK2), signal transducer and activator of transcription (STAT) 1α, MAP kinase kinase (MEK1/2), extracellular signal-regulated kinases 1 and 2 (Erk1/Erk2) and nuclear factor kappa B (NF-κB) – in the regulation of NO production by IFN-γ-stimulated J774 murine Mφ. The use of specific signalling inhibitors permitted us to establish that JAK2/STAT1α- and Erk1/Erk2-dependent pathways are the main players in IFN-γ-inducible Mφ NO generation. To determine whether the inhibitory effect was taking place at the pre- and/or post-transcriptional level, we evaluated the effect of each antagonist on inducible nitric oxide synthase (iNOS) gene and protein expression, and on the capacity of IFN-γ to induce JAK2, Erk1/Erk2 and STAT1α phosphorylation. All downregulatory effects occurred at the pretranscriptional level, except for NF-κB, which seems to exert its role in NO production through an iNOS-independent event. In addition, electrophoretic mobility shift assay (EMSA) analysis revealed that STAT1α is essential for IFN-γ-inducible iNOS expression and NO production, whereas the contribution of NF-κB to this cellular regulation seems to be minimal. Moreover, our data suggest that Erk1/Erk2 are responsible for STAT1α Ser727 residue phosphorylation in IFN-γ-stimulated Mφ, thus contributing to the full activation of STAT1α. Taken together, our results indicate that JAK2, MEK1/2, Erk1/Erk2 and STAT1α are key players in the IFN-γ-inducible generation of NO by Mφ.