We established a method for fully automated measurement of total iron-binding capacity (TIBC) in serum without separation of the unbound excess iron after saturating serum transferrin. After saturation of serum transferrin with an excess amount of iron (first step), the unbound iron was eliminated by formation of a complex with ferrozine, which was used as a chromogenic reagent (second step). For the TIBC assay, iron dissociated from transferrin by shifting the pH to acidic was reacted with ferrozine, and the increase in the absorbance at 570 nm was measured (third step). Because the iron used as a calibrator, which was added to saturate transferrin, reacted completely with ferrozine in the second step (elimination of unbound iron), the change in the absorbance to generate a calibration factor could not be monitored in the third step. To solve this problem, we used N-(2-hydroxyethyl)ethylenediamine-N,N',N'-triacetic acid (HEDTA) to complex with the iron added to saturate transferrin in the second step. This made it possible to form an iron-ferrozine complex at acidic pH because iron was dissociated from HEDTA at acidic pH. The within-run CVs of this method were 0.66-2.43% at 17.7-77.0 mumol/L, and the day-to-day CVs were 1.06-1.57% at 29.9-60.4 mumol/L (n = 10). The correlation between the values obtained with this method (y) and those from the direct TIBC assay, which involved removal of unbound iron by ion-exchange resin (x), was: y = 0.963x + 0.29 mumol/L (r = 0.973, Sy/x = 2.83, n = 59), and with the TIBC values calculated from the serum iron concentrations and the unbound iron-binding capacities measured by a direct colorimetric method (x) was: y = 1.01x - 1.06 mumol/L (r = 0.994, Sy/x = 1.66, n = 51).