The process of iron (Fe) release from cells plays an important role in health and disease, although the mechanisms responsible remain unclear. In this study we have examined the process of Fe efflux from HepG2 cells, including the possible roles of Cp and ascorbate in this process. Recently, it has been suggested that Cp plays no role in Fe release but can increase Fe uptake by Fe-deficient HepG2 cells (Mukhopadhyay et al. Science 1998;279:714-7). However, this latter study used a nonphysiologically relevant Fe complex (iron 59-NTA) to label cells with 59Fe at a nonphysiologic temperature (25 degrees C) and Cp concentration (<100 microg/mL). Because of these problems, the experiments have been repeated by maintaining physiologic conditions and labeling cells with the physiologic Fe donor diferric Tf. When cells were labeled at 37 degrees C with 59Fe-Tf in the presence of a physiologically relevant Cp concentration (300 microg/mL), this latter protein had no effect on the uptake of 59Fe in control cells or in cells depleted of Fe by using desferrioxamine. In addition, when Fe-replete or Fe-depleted cells were incubated with 59Fe-NTA at 25 degrees C or 37 degrees C, Cp had no effect on 59Fe uptake compared with the control. When the effect of Cp (10-500 microg/mL) on 59Fe release was examined in cells prelabeled with 59Fe-Tf, a concentration-dependent increase in 59Fe efflux was observed, whereas BSA had no effect. However, in contrast to membrane-permeable Fe chelators that caused a marked increase in Fe release, the effect of Cp on Fe efflux was less impressive. To further investigate the mechanism of 59Fe mobilization, we compared 59Fe efflux among HepG2 cells, SK-Mel-28 melanoma cells, and SK-N-MC neuroblastoma cells. These studies demonstrated that 59Fe release was dependent on the incubation time with 59Fe-Tf, the cell line, and the reincubation temperature. Although 59Fe mobilization from cells was markedly temperature dependent, a range of metabolic inhibitors did not affect 59Fe release. Additional experiments showed that physiologic concentrations of ascorbate reduced 59Fe efflux, whereas glutathione had no effect. This study provides further evidence that Cp is involved in Fe mobilization but does not appear to affect Fe uptake from Tf or NTA.