Transferrin, the serum Fe-transport protein which can bind 2 metal ions at physiologic pH, binds just one Fe3+, VO2+ or Cr3+ at pH 6.0. Fe3+ and VO2+ appear to be bound at the same site, designated A, based on EPR spectra of VO2+-transferrin and (Fe3+)1(VO2+)1-transferrin. The EPR spectra of (Cr3+)1(VO2+)1-transferrin and of (Cr3+)1(Fe3+)1-transferrin indicate that Cr3+ is bound to site B at pH 6.0. Transferrin was labeled at site A with 59Fe at pH 6.0 and at site B with 55Fe at pH 7.5. When the pH of the resulting preparation was lowered to 6.3 and the dissociated iron was separated by gel filtration, about 10 times as much 55Fe as 59Fe was lost. The same EPR and isotopic-labeling experiments showed that Fe3+ added to transferrin at pH 7.5 binds to site A with about 90% selectivity.