Sepharose beads bound to 55Fe-transferrin (Tf) were used to evaluate Tf-dependent iron uptake not employing receptor-mediated endocytosis (RME). The iron of 55Fe2-Tf-Sepharose was reduced and taken up by cultured human fibroblasts in a time- and concentration-dependent fashion (Km 7 microM; Vmax 128 pmol/mg/min). This redox system resembled that for Tf-independent iron uptake (Tf-IU, evaluated using 55Fe-citrate) in several ways. 1) NH4Cl did not inhibit iron uptake from 55Fe-citrate and 55Fe2-Tf-Sepharose but did inhibit uptake from 55Fe2-Tf (RME system). 2) Iron uptake and reduction from 55Fe2-Tf-Sepharose and 55Fe-citrate increased with temperature hyperbolically, differing from the sigmoidal curve for RME uptake. 3) The subcellular distributions of iron from 55Fe-citrate and 55Fe2-Tf-Sepharose resembled each other and differed from that for 55Fe2-Tf. 4) The optimal pH for iron reduction and uptake using 55Fe2-Tf-Sepharose or 55Fe-citrate was less than pH 5.5, while that for iron uptake from 55Fe2-Tf was pH 7.4. 5) The uptake and reduction of iron from 55Fe2-Tf-Sepharose was inhibited by ferric citrate and by transition metals. We conclude that both Tf-independent and non-RME, Tf-dependent iron uptake proceed via a common redox system for iron. The mechanisms of cellular iron uptake can be separately evaluated in fibroblasts using 55Fe-citrate, 55Fe2-Tf, and 55Fe2-Tf-Sepharose beads.