Competitive binding of Fe(3+), Cr(3+), and Ni(2+) to transferrin (Tf) was investigated at various physiological iron to Tf concentration ratios. Loading percentages for these metal ions are based on a two M(n+) to one Tf (i.e., 100% loading) stoichiometry and were determined using a particle beam/hollow cathode-optical emission spectroscopy (PB/HC-OES) method. Serum iron concentrations typically found in normal, iron-deficient, iron-deficient from chronic disease, iron-deficient from inflammation, and iron-overload conditions were used to determine the effects of iron concentration on iron loading into Tf. The PB/HC-OES method allows the monitoring of metal ions in competition with Fe(3+) for Tf binding. Iron-overload concentrations impeded the ability of chromium (15.0 μM) or nickel (10.3 μM) to load completely into Tf. Low Fe(3+) uptake by Tf under iron-deficient or chronic disease iron concentrations limited Ni(2+) loading into Tf. Competitive binding kinetic studies were performed with Fe(3+), Cr(3+), and Ni(2+) to determine percentages of metal ion uptake into Tf as a function of time. The initial rates of Fe(3+) loading increased in the presence of nickel or chromium, with maximal Fe(3+) loading into Tf in all cases reaching approximately 24%. Addition of Cr(3+) to 50% preloaded Fe(3+)-Tf showed that excess chromium (15.0 μM) displaced roughly 13% of Fe(3+) from Tf, resulting in 7.6 ± 1.3% Cr(3+) loading of Tf. The PB/HC-OES method provides the ability to monitor multiple metal ions competing for Tf binding and will help to understand metal competition for Tf binding.