Samples of human Fe∙transferrin (Fe∙HTr) were prepared from a single batch of apotransferrin (apo∙HTr) by either the Fe(III)–citrate or the Fe(II)–ceruloplasmin (ferroxidase) method. By using 55Fe, 55Fe∙HTr prepared by the citrate method and 55Fe∙HTr prepared by the ceruloplasmin method contained 2.2–2.3 and 2.0 Fe/mol, respectively. For both 55Fe∙HTr preparations, the isotope was shown to be associated with the protein from the measurement of absorbance at 465 nm and dialysis studies. However, passage of the 55Fe∙HTr (ceruloplasmin) reaction mixture through DEAE-cellulose caused 55–60% of 55Fe to be lost from the protein, although no decrease in absorbance at 465 nm was observed. Ion-exchange chromatography of 55Fe∙HTr (citrate) did not induce loss of 55Fe. Absorbance measurements showed significant differences between the two Fe∙HTr preparations with respect to the ratios A212/A278 and A463/A278. Using an excitation wavelength of 275 nm, the fluorescence intensity ratios relative to apo∙HTr were 0.275 and 0.309 for Fe∙HTr (citrate) and Fe∙HTr (ceruloplasmin), respectively. Electron spin resonance (ESR) measurements confirmed that Fe∙HTr (citrate) and Fe∙HTr (ceruloplasmin) were saturated with Fe. Hyperfine coupling constants and other features of the resonance profile revealed distinct differences between the two Fe∙HTr preparations. Dialysis against H2O caused Fe∙HTr (citrate), but not Fe∙HTr (ceruloplasmin), to lose absorbance at 465 nm. The ESR profile of Fe∙HTr (citrate), after dialysis against H2O, was reduced to multiple splittings and a lack of resolution of the central hyperfine structure. Addition of Na2CO3 restored the absorbance (465 nm) and the ESR pattern of Fe∙HTr (citrate). In contrast, these properties of Fe∙HTr (ceruloplasmin) were little affected by dialysis against H2O. However, the addition of trisodium citrate to Fe∙HTr (ceruloplasmin) caused a reduction in absorbance at 465 nm and a change in ESR profile to resemble that of Fe∙HTr (citrate) after dialysis in H2O; these changes, caused by citrate binding to Fe∙HTr (ceruloplasmin), were restored to normal by the addition of Na2CO3. The data indicate that different protein conformations result from complexing Fe(III) with apo∙HTr by these two different procedures. The two Fe∙HTr products may differ, conceivably, in their abilities to transfer Fe to cells.