Bovine and possum macrophages were infected in vitro with a virulent strain of Mycobacterium bovis, and mycobacterial replication was measured in the infected macrophages cultured under a variety of conditions. Virulent M. bovis replicated substantially in alveolar possum macrophages as well as in bovine blood monocyte-derived macrophages. Addition of recombinant bovine interferon-gamma (IFN-gamma) with low concentrations of lipopolysaccharide (LPS) rendered bovine macrophages significantly more resistant to M. bovis replication. Disruption of iron levels in infected macrophages by addition of apotransferrin or bovine lactoferrin blocked replication of M. bovis in both bovine and possum macrophages. On the other hand, addition of exogenous iron, either in the form of iron citrate or iron-saturated transferrin, rendered macrophages of both species much more permissive for the replication of M. bovis. The impact of iron deprivation/loading on the mycobacteriostatic activity of cells was independent of nitric-oxide release, as well as independent of the generation of oxygen radical species in both possum and bovine macrophages. Exogenous iron was shown to reverse the ability of IFN-gamma/LPS pulsed bovine macrophages to restrict M. bovis replication. When autologous possum lymphocytes from animals vaccinated with M. bovis strain BCG were added to infected macrophages, they rendered the macrophages less permissive for virulent M. bovis replication. Loading the cells with iron prior to this macrophage-lymphocyte interaction, reversed this immune effect induced by sensitized cells. We conclude that, in two important animal species, intracellular iron level plays an important role in M. bovis replication in macrophages, irrespective of their activation status.