MntH, a bacterial homolog of the mammalian natural resistance-associated macrophage protein 1 (Nramp1), is a primary Mn(2+) transporter of Salmonella enterica serovar Typhimurium and Escherichia coli. S. enterica serovar Typhimurium MntH expression is important for full virulence; however, strains carrying an mntH deletion are only partially attenuated and display no obvious signs of Mn(2+) deficiency. We noted that promoter sequences for mntH and for the putative Fe(2+) transporter sitABCD appeared to have the same regulatory element responsive to Mn(2+) and so hypothesized that sitABCD could transport Mn(2+) with high affinity. We have now characterized transport by SitABCD in S. enterica serovar Typhimurium using (54)Mn(2+) and (55)Fe(2+) and compared its properties to those of MntH. SitABCD mediates the influx of Mn(2+) with an apparent affinity (K(a)) identical to that of MntH, 0.1 microM. It also transports Fe(2+) but with a K(a) 30 to 100 times lower, 3 to 10 microM. Inhibition of (54)Mn(2+) transport by Fe(2+) and of (55)Fe(2+) transport by Mn(2+) gave inhibition constants comparable to each cation's K(a) for influx. Since micromolar concentrations of free Fe(2+) are improbable in a biological system, we conclude that SitABCD functions physiologically as a Mn(2+) transporter. The cation inhibition profiles of SitABCD and MntH are surprisingly similar for two structurally and energetically unrelated transporters, with a Cd(2+) K(i) of approximately 1 microM and a Co(2+) K(i) of approximately 20 microM and with Ni(2+), Cu(2+), and Fe(3+) inhibiting both transporters only at concentrations of >0.1 mM. The one difference is that Zn(2+) exhibits potent inhibition of SitABCD (K(i) = 1 to 3 microM) but inhibits MntH weakly (K(i) > 50 microM). We have previously shown that MntH transports Mn(2+) most effectively under acidic conditions. In sharp contrast, SitABCD has almost no transport capacity at acid pHs and optimally transports Mn(2+) at slightly alkaline pHs. Overall, coupled with evidence that each transporter is multiply but distinctly regulated at the transcriptional level, the distinct transport properties of MntH versus SitABCD suggest that each transporter may be specialized for Mn(2+) uptake in different physiological environments.