The kinetics of binding of Cu(II), Tb(III) and Fe(III) to ovotransferrin have been investigated using the stopped-flow technique. Rate constants for the second-order reaction, k+, were determined by monitoring the absorbance change upon formation of the metal-transferrin complex in time range of milliseconds to seconds. The N and C sites appeared to bind a particular metal ion with the same rate; thus, average formation rate constants k+ (average) were 2.4 x 10(4) M-1 s-1 and 8.3 x 10(4) M-1 s-1 for Cu(II) and Tb(III) respectively. Site preference (N site for Cu(II) and C site for Tb(III] is then mainly due to the difference in dissociation rate constant for the metals. Fe(III) binding from Fe-nitrilotriacetate complex to apo-ovotransferrin was found to be more rapid, giving an average formation rate constant k+ (average) of 5 x 10(5) M-1 s-1, which was followed by a slow increase in absorbance at 465 nm. This slow process has an apparent rate constant in the range 3 s-1 to 0.5 s-1, depending upon the degree of Fe(III) saturation. The variation in the rate of the second phase is thought to reflect the difference in the rate of a conformational change for monoferric and diferric ovotransferrins. Monoferric ovotransferrin changes its conformation more rapidly (3.4 s-1) than diferric ovotransferrin (0.52 s-1). A further absorbance decrease was observed over a period of several minutes; this could be assigned to release of NTA from the complex, as suggested by Honda et al. (1980).