In the present study we analysed the mechanism of intracellular routing of iron acquired by erythroid cells via receptor-mediated endocytosis of Tf-Fe [Tf (transferrin)-iron]. Using real-time fluorimetry and flow cytometry, in conjunction with targeted fluorescent metal sensors, we monitored concurrently the cytosolic and mitochondrial changes in labile iron evoked by endocytosed Tf-Fe. In K562 human erythroleukaemia cells, most of the Tf-Fe was found to be delivered to the cytosolic labile iron pool by a saturable mechanism [60-120 nM Km (app)] that was quantitatively dependent on: Tf receptor levels, endosomal acidification/reduction for dislodging iron from Tf and ensuing translocation of labile iron into the cytosolic compartment. The parallel ingress of iron to mitochondria was also saturable, but with a relatively lower Km (app) (26-42 nM) and a lower maximal ingress per cell than into the cytosol. The ingress of iron into the mitochondrial labile iron pool was blocked by cytosol-targeted iron chelators, implying that a substantial fraction of Tf-Fe delivered to these organelles passes through the cytosol in non-occluded forms that remain accessible to high-affinity ligands. The present paper is the first report describing intracellular iron routing measured in intact cells in real-time and in quantitative terms, opening the road for also exploring the process in mixed-cell populations of erythroid origin.