The potential roles of vesicular transport and apotransferrin (entering from the blood) in intestinal Fe absorption were investigated using Caco2 cell monolayers with tight junctions in bicameral chambers as a model. As shown previously, addition of 39 µM apotransferrin (apoTf) to the basolateral fluid during absorption studies markedly stimulated overall transport of 1 µM ⁵⁹Fe from the apical to the basal chamber, and stimulated its basolateral release from prelabeled cells, implicating endo and exocytosis. Rates of transport more than doubled. Uptake was also stimulated, but only 20%. Specific inhibitors of aspects of vesicular trafficking were applied to determine their potential effects on uptake, retention and basolateral (overall) transport of ⁵⁹Fe. Nocodazole and 5'-(4-fluorosulfonylbenzoyl)-adenosine each reduced uptake and basolateral transport up to 50%. Brefeldin A inhibited about 10%. Tyrphostin A8 (AG10) reduced uptake 35% but markedly stimulated basolateral efflux, particularly that dependent upon apoTf. Cooling of cells to 4^o (which causes depolymerization of microtubules and lowers energy availability) profoundly inhibited uptake and basolateral transfer of Fe (7-12-fold). Apical efflux (which was substantial) was not temperature-affected. Our results support the involvement of apoTf cycling in intestinal Fe absorption and indicate that as much as half of the iron uses apoTf and non-apoTf-dependent vesicular pathways to cross the basolateral membrane and brush border of enterocytes.