We recently reported that the infection of macrophages with Leishmania major led to the release of type 1 interferons (IFN-alpha /beta ). Moreover, at day 1 of infection of mice with L. major, IFN-alpha /beta was required for the expression of type 2 (inducible) NO synthase (NOS2 or iNOS) which, however, was restricted to a few macrophages in the dermis. Here, we further characterized the regulation of NOS2 by IFN-alpha /beta. Macrophages that were either simultaneously or sequentially exposed to L. major promastigotes and IFN-alpha /beta expressed NOS2 and anti-leishmanial activity. In contrast, when high amounts of IFN-alpha /beta were used or when IFN-alpha /beta was added to the macrophages 2 h prior to the parasites, almost no induction of NOS2 was observed. After pretreatment with IFN-alpha /beta, tyrosine phosphorylation and nuclear DNA binding of Stat1alpha, the degradation of the NF-kappaB inhibitor (IkappaBalpha and beta), and the nuclear translocation of NF-kappaB were strongly impaired compared with macrophages exposed to IFN-alpha /beta and L. major simultaneously. Thus, IFN-alpha /beta exerts agonistic or antagonistic effects on the expression of NOS2 in macrophages infected with a microbial pathogen, depending on the sequence of the stimuli and the amount of IFN-alpha /beta added. The limited number of NOS2-positive macrophages at day 1 of infection in vivo might result from a blockage of non-infected macrophages by IFN-alpha /beta that is released by neighboring infected cells.