The possibilities that the recycling of the transferrin receptor is a rate-limiting step in the efflux of endocytosed transferrin, and that the receptor functions as a trans-membrane Fe transporter were investigated in untransfected Ltk- cells and in cells transfected with different levels of DNA for wild-type, mutant and chimeric human transferrin receptors. The uptake of transferrin-bound Fe and non-transferrin-bound Fe(II), and the surface binding, endocytosis and recycling of transferrin were measured. In cells that expressed increasing numbers of surface transferrin receptors, the rate of Fe uptake increased at a slower rate than the number of receptors. By measurement of the rates of endocytosis and recycling of transferrin it was shown that this effect was not due to a deficiency of endocytosis, but to a slower rate of recycling as the receptor numbers increased. Hence, a restricted recycling rate of the transferrin receptor appeared to be responsible for the slower rate of Fe uptake by cells with high receptor numbers, presumably because one or more cytosolic components required for recycling were in limited supply. The rate of uptake of non-transferrin-bound Fe(II) was not influenced by the number of transferrin receptors present on the surface of the cells even though this varied more than 20-fold between the different cell lines. Hence, this investigation does not support the hypothesis that the receptors play a direct role in the transport of Fe(II) across cell membranes, as has been proposed previously [Singer, S. J. (1989) Biol. Cell 65, 1-5].