The present study characterizes the transport of nontransferrin (non-Tf) iron by K562 cells. Accumulation of radiolabel by cells incubated with 55Fe-nitrilotriacetate (NTA) is a saturable process that is time and temperature dependent (Ea approximately 20 kcal/mol). Initial rate analysis of iron influx yields values of Vmax = 855 fmol/min/10(6) cells and apparent Km = 0.54 microM. NHCL4 and chloroquine, agents that block cellular acquisition of iron from Tf, do not interfere with assimilation from FeNTA, demonstrating that uptake is truly independent of the Tf-mediated pathway. Furthermore, the inactivation of this transport mechanism by limited proteolytic digestion on ice indicates that specific cell surface proteins are involved. The extent of radiolabel incorporation into heme and ferritin is the same regardless of whether K562 cells acquire iron from 55FeNTA via the cell surface mechanism or from 55Fe-Tf via receptor-mediated endocytosis. Unlike other Tf-independent iron transport pathways that have been described, the K562 cell transport mechanism is not inhibited by divalent cations such as Ni2+, Co2+, or Mn2+. Uptake from 55FeNTA can be blocked by Cu2+ but at concentrations > 1500-fold molar excess. However, Cd2+ is a fairly specific inhibitor of 55Fe uptake by K562 cells (IC50 approximately 50 microM). Additionally, the K562 cell transport mechanism is not Ca2+ dependent and does not appear to be regulated by extracellular iron salts, in contrast to features noted for non-Tf iron uptake by fibroblasts (Sturrock, A., Alexander, J., Lamb, J., Craven, C. M., and Kaplan, J. (1991) J. Biol. Chem. 265, 3139-3145; Kaplan, J., Jordan, I., and Sturrock, A. (1991) J. Biol. Chem. 266, 2997-3004). These unique characteristics of the K562 cell uptake mechanism suggest that multiple transport systems function in Tf-independent iron assimilation.