Classically, the iron binding protein transferrin (Tf) is responsible for delivery of iron into the majority of cell types. Receptor-mediated endocytosis of holo-transferrin into cells is via the ubiquitously expressed transferrin receptor 1 (TfR1). However, some cell types, including enterocytes, glial cells, placental cytotrophoblasts and kidney proximal tubule epithelia, also express the megalin/cubilin receptor complex, that has affinity for Tf and thus offer an alternative means of receptor mediated cellular Tf uptake. Multi-ligand receptors, megalin/cubilin are suggested to be the mediators of Tf uptake in the proximal tubule (Kozyraki, et al. 2001). However in mice, the TfR1 has been shown to localise at the apical membrane of the proximal tubule epithelia (Zhang, et al. 2007). The aim of this study was to determine the role of TfR1 and megalin/cubilin complex to cellular transferrin delivery. Experiments were performed in vitro, using a cell line (WKPT-0293 Cl.2 (WKPT)) derived from the proximal tubule of rats. Ligands of megalin/cubilin, receptor associated protein (RAP) and Tf were used for uptake studies. RT-PCR showed that WKPT cells contain mRNA transcripts encoding TfR1 and megalin. Furthermore under iron restriction (100μM desferrioxamine (DFO)) TfR1 mRNA levels increased and megalin mRNA decreased after 40hr of iron restriction. Conversely, iron enrichment (100μM ferric ammonium citrate) decreased TfR mRNA and elevated megalin mRNA levels. Ligand uptake studies showed that under control conditions dsRED conjugated RAP (dsRED-RAP) and alexa488 conjugated Tf (Tf-488) were internalised. In control cells, pre-binding of unconjugated RAP (his-RAP), inhibited dsRED-RAP and Tf-488 uptake, suggesting megalin/cubilin to be the predominant mechanism of Tf uptake under normal conditions. Under iron restriction, uptake of Tf-488 markedly increased in comparison to control treated cells, whilst no qualitative change in dsRED-RAP uptake was observed. Pre-binding of his-RAP inhibited most dsRED-RAP uptake. Interestingly, pre-binding of his-RAP had no detectable effect on the uptake of Tf-488, suggesting that TfR1 is the primary mechanism of Tf uptake under iron restriction. These data strongly suggests that megalin and TfR1 are differentially regulated by iron. Furthermore, megalin/cubilin under iron replete conditions are the dominant means by which Tf is endocytosed. However, upon iron restriction, TfR1 switches to become the principal receptor for Tf endocytosis. These data potentially provide an insight in to the interplay of transferrin binding receptors in the control of cellular iron balance.