The roles of the genes feoB (ABC ferrous iron transporter), mntH (proton-dependent manganese transporter), and sitABCD (putative ABC iron and/or manganese transporter) in Salmonella pathogenicity were investigated by using mutant strains deficient in one, two, or three transporters. Our results indicated that sitABCD encodes an important transporter of Mn(II) and Fe(II) which is required for full virulence in susceptible animals (Nramp1(-/-)) and for replication inside Nramp1(-/-) macrophages in vitro. The mntH sitABCD double mutant (mutant MS) showed minimal Mn(II) uptake and increased sensitivity to H(2)O(2) and to the divalent metal chelator 2,2'-dipyridyl (DP) and was defective for replication in macrophages. In vivo MS appeared to be as virulent as the sitABCD mutant in Nramp1(-/-) animals. The ferrous iron transporter Feo was required for full virulence in 129/Sv Nramp1(-/-) mice, and infection with multiple mutants lacking FeoB was not fatal. The sitABCD feoB mutant (mutant SF) and the mntH sitABCD feoB mutant (mutant MSF) showed minimal Fe(II) uptake and were slightly impaired for replication in susceptible macrophages. MSF showed reduced growth in minimal medium deficient in divalent cations. The role of the mntH gene, which is homologous to mammalian Nramp genes, was also investigated after overexpression in the double mutant MS. MntH preferred Mn(II) over Fe(II) and could suppress MS sensitivity to H(2)O(2) and to DP, and it also improved the intracellular survival in Nramp1(-/-) macrophages. This study indicates that acquisition of Mn(II), in addition to Fe(II), is required for intracellular survival and replication of Salmonella enterica serovar Typhimurium in macrophages in vitro and for virulence in vivo.