Caco-2 cells grown in bicameral chambers, a model of intestinal epithelial iron transport (Biochim. Biophys. Acta (1991) 1070, 205-208), were used to study the effect of apo-transferrin (apo-Tf) in the basal chamber on 59Fe uptake from the apical surface, intracellular 59Fe distribution, and 59Fe transport into the basal chamber. Caco-2 cells were grown with varying amounts of iron to achieve cells that were either iron-deficient (FeD), or normal iron status (FeN), or iron-loaded (FeH). The effect of apo-Tf was most marked in FeD cells with the transport of 59Fe from 1 microM 59Fe-ascorbate on the apical side to the basal chamber measured as (22.2 +/- 3.0) x 10(4), (8.2 +/- 0.6) x 10(4), and (2.7 +/- 0.4) x 10(4) atoms 59Fe/cell/min in the presence of apo-Tf, BSA, and no added protein, respectively. Unexpectedly in FeD cells total 59Fe uptake (i.e., both 59Fe in the cells and that transported into the basal chamber) was decreased by basolateral apo-Tf with total uptake of (2.6 +/- 0.3) x 10(5), (4.8 +/- 0.6) x 10(5), and (4.8 +/- 0.7) x 10(5) atoms/cell/min with apo-Tf, BSA, and no additions, respectively. Analysis of intracellular 59Fe by isoelectrofocusing in polyacrylamide gels demonstrated 59Fe migrating both with a basic pI and with the pI values of ferritin (Ft) at a ratio of 200:1 (basic pI moiety: ferritin) in FeD cells. The presence of Tf further decreased the small amount of 59Fe in Ft. These studies demonstrate that basolateral Tf affects the apical uptake of 59Fe, the intracellular distribution of 59Fe, and the transport of 59Fe across intestinal epithelium, the latter effect occurring even when cellular content of ferritin is high.