Samples of human apotransferrin (apo∙HTr) were saturated with Fe(III) by two different techniques, a method employing excess trisodium citrate to chelate Fe(III) and a nonchelating approach which involves the ferroxidase activity of ceruloplasmin to convert Fe(II) → Fe(III). The samples were radiolabelled with either 55Fe or 3H. Using an initial molar Fe/apo∙HTr ratio of 2.0–2.1, preparations of human transferrin with bound Fe (Fe∙HTr) using the citrate method invariably contained 2.2–2.4 atoms Fe/molecule, whereas Fe∙HTr (ceruloplasmin method) contained 2.0 atoms/molecule as shown by spectrophotometric and radioactivity measurements. Uptake of Fe from these Fe∙HTr preparations by K-562 cells grown in a serum-free medium was marginally, but consistently, more rapid from 55Fe∙HTr (citrate) than from 55Fe∙HTr (ceruloplasmin). Taking account of the different Fe contents of the Fe∙HTr preparations, the rate measured over a 2-h period amounted to approximately 12 700 and 16 100 Fe atoms/(cell∙min) for Fe∙HTr (ceruloplasmin) and Fe∙HTr (citrate), respectively. However, cell binding by the two Fe∙[3H]HTr preparations did not differ significantly over the 8-h incubation period. Furthermore, from the 3H distribution, the quantities of Fe∙HTr bound reversibly at the cell surface and contained within the cell were similar for the two Fe∙HTr preparations. The results indicate that apo∙HTr may bind Fe in different ways depending on the method of Fe presentation and that the Fe∙HTr product can donate Fe to K∙562 cells at a rate which may reflect the method used for Fe-complex formation.