Nramp1 controls responses to infection and encodes a biallelic (G169D) macrophage-restricted divalent-cation transporter. Nramp1(D169) is phenotypically null. We demonstrate Nramp1 is implicated in iron regulation in vivo. In spleen, expression is exclusive to Nramp1(G169) strains within the red pulp. By morphometric analysis, the distribution of splenic iron, following systemic overload, correlates with Nramp1 genotype. More iron is located within the red pulp in Nramp1(D169) strains, whereas in Nramp1(G169) strains iron deposits are localized within the marginal-zone metallophilic cells. Nramp1 immunoreactive protein is not present in control brain, but inducible within a hemorrhagic lesion model in Nramp1(G169) strains. Nramp1 protein expression demonstrates an inverse correlation to the presence of iron. Nramp1(G169) strains show no Perl's stain-reactive iron within the lesion. In contrast, Nramp1(D169) strains display iron-staining cells. The process of cellular iron regulation was investigated in vitro in Nramp1(G169) transfectant Raw264.7 macrophages. Greater (30-50%) iron efflux from Nramp1(G169) compared with Nramp1(D169) cells was determined. The extent of Nramp1-dependent iron-release was influenced by bafilomycin A1, and endogenous nitric oxide synthesis, both inhibitors of vacuolar-ATPase. This study demonstrates that Nramp1 regulates macrophage iron handling, and probably facilitates iron release from macrophages undergoing erythrophagocytosis in vivo.