Differentiated Caco-2 colon carcinoma cell monolayers grown in bicameral chambers have been used as an in vitro model to study the effect of different carrier molecules on mucosal iron transport. Transfer of iron across the monolayers in the apical-to-basolateral direction was greater from ferric lactoferrin than from iron citrate, while very little transport occurred from Fe-transferrin. However, a greater proportion of iron was retained by the cells when Fe-citrate was the donor. Caco-2 cells expressed transferrin receptors (n = 1.3 x 10(5) /cell; Ka = 2 x 10(8) l/mol), but binding of lactoferrin, though substantial in quantity, had an affinity too low to measure. When monolayers were incubated with 125I-labelled lactoferrin or transferrin some 125I-activity was transported, but almost all was TCA-soluble, suggesting that degradation products rather than intact protein were being transported. Addition of 10 microM S-nitroso-N-acetyl-D,L-penicillamine (SNAP), which produces nitric oxide (NO) in solution, caused a significant increase in iron transport from ferric citrate, but not from Fe-lactoferrin or Fe-transferrin. It is concluded that in this in vitro system lactoferrin but not transferrin enhances mucosal iron transport, and that NO may play a regulatory role in iron absorption.