The receptor-mediated endocytotic cycle of rat and human transferrin has been studied in intact, isolated rat seminiferous tubules and Sertoli cells in culture. Double-labeled [( 59Fe125I]) transferrin has been used to study the fate of transferrin and iron. Diferric transferrin binds to the tubules and the cultured Sertoli cells and is internalized. The iron remains inside, while the transferrin recycles and is released into the medium. Although, as reported before (Wauben-Penris et al., 1986), "extra" binding sites for human transferrin exist as compared to rat transferrin, this does not result in extra uptake of transferrin or iron. Both rat and human transferrin transport iron into the cells and recycle back to the surface, and do so with identical kinetics. A striking difference has been found between the mean efficient recycling times of the transferrin receptors in intact tubules (90 min) and in Sertoli cells in culture (21 min). Possible explanations of this difference are discussed. Light-microscopic autoradiography of [125 I]-labeled transferrin has revealed that the transferrin protein is excluded from the adluminal compartment, even after 21 h of incubation. This indicates that externally added transferrin itself does not deliver iron to the postmeiotic germ cells in intact, isolated rat seminiferous tubules.