The serum albumin is the most abundant protein in blood plasma and the iron is essential for many cellular processes. However, the interaction between Fe(3+) and haem-free serum albumin remains unclear. Here we provide evidence for the fact that haem-free BSA possesses one specific Fe(3+)-binding site. The binding of Fe(3+) to BSA results in a significant quenching of the Trp fluorescence of BSA. The average apparent dissociation constant value for the interaction of Fe(3+) and BSA is 3.46 x 10(-8)+/-3 x 10(-10) M at 37 degrees C and 3.30 x 10(-8)+/-5 x 10(-10) M at 25 degrees C, respectively, as determined by fluorescence titration. Addition of 50 microM Fe(2+) to 1 microM BSA results in an obvious hysteretic effect on the fluorescence of BSA. The time-dependent fluorescence quenching of BSA by Fe(2+) is not caused by the Fe(2+)-induced conformational change of BSA, but the oxygen-dependent oxidation of Fe(2+) to Fe(3+). Fe(2+) undergoes an oxygen-dependent oxidation to Fe(3+) under aerobic conditions, which is accelerated by the interaction of BSA with Fe(3+) and extensively inhibited under anaerobic conditions. The results suggest that BSA may take part in non-transferrin bound iron transfer.