We previously developed the highly sensitive perfusion-Perls and -Turnbull methods to visualize nonheme ferric (Fe (III)) and ferrous (Fe (II)) iron, respectively. The present study used these methods to investigate the possible presence of nonheme iron in the redox (ferric/ferrous) state in the noneheme iron store (phagolysosomes and siderosomes) of resident macrophages in the rat. The perfusion-Perls and -Turnbull methods at pH 0.6 supplemented by DAB intensification intensely stained resident macrophages of different tissues and organs of normal and iron-overloaded rats. The perfusion-Turnbull method, which is specific for nonheme Fe (II), partly stained hemosiderin at pH 5.3, but hardly stained it at the physiological pH, suggesting the presence of some iron in the reduced form, free Fe2+ and/or loosely bound Fe (II), at the intravacuolar pH (5.4+/-0.2) of the phagolysosomes of macrophages. Electron microscopy of the splenic and hepatic macrophages treated by the perfusion-Perls or -Turnbull method showed that Fe (II) deposits were largely distributed along the margin of hemosiderin masses while Fe (III) deposits could also be found within hemosiderin masses.