A/J mouse macrophages infected with Legionella pneumophila and treated with gamma interferon (IFN-gamma) in vitro developed potent antimicrobial activity. This antilegionella activity was independent of the macrophage capacity to generate reactive oxygen intermediates, since the oxygen radical scavengers catalase, superoxide dismutase, mannitol, and thiourea had no effect on the antilegionella activity of IFN-gamma-activated macrophages. Likewise, whereas the ability of IFN-gamma-activated macrophages to synthesize reactive nitrogen intermediates was markedly inhibited by the L-arginine (Arg) analogs, NG-monomethyl-L-arginine and L-aminoguanidine, as well as by incubation in L-Arg-free medium, their ability to inhibit the intracellular growth of L. pneumophila remained intact. The intracellular growth of L. pneumophila in A/J macrophages was inhibited by the iron(III) chelator desferrioxamine and reversed by Fe-transferrin as well as by ferric salts. Additionally, IFN-gamma-activated macrophages incorporated 28% less 59Fe(III) compared with nonactivated cells. Nonetheless, only partial blocking of growth restriction was observed when IFN-gamma-stimulated macrophages were saturated with iron(III). Indole-propionic acid, which appears to inhibit the biosynthesis of L-tryptophan (L-Trp), was an L-Trp-reversible growth inhibitor of L. pneumophila in macrophages, implying that the intracellular replication of this pathogen is also L-Trp dependent. However, an excess of exogenous L-Trp did not reverse the growth inhibition due to IFN-gamma, though a small synergistic effect was observed when the culture medium was supplemented with both iron(III) and L-Trp. We conclude that IFN-gamma-activated macrophages inhibit the intracellular proliferation of L. pneumophila by reactive oxygen intermediate- and reactive nitrogen intermediate-independent mechanisms and just partially by nutritionally dependent mechanisms. We also suggest that additional mechanisms, still unclear, may be involved, since complete reversion was never obtained and since at higher concentrations of IFN-gamma, iron(III) did not induce any significant reversion in the L. pneumophila growth inhibition.