The second-order rate constants for iron removal from citrate by the chelating agents desferrioxamine B (DFO) and 3-hydroxy-1,2-dimethyl-4-pyridone (L1) were determined. The overall rate constant k(on) for the transfer of Fe from citrate to the chelator is 4 M(-1) s(-1) for DFO and 43 M(-1) s(-1) for L1 at pH 7.4 and 37 degrees C. The kinetics of transfer of iron from citrate to the two chelators was examined in detail, and a possible mechanism is proposed. Both DFO and L1 form a mixed complex with citrate-iron prior to the transfer of the metal ion. The dissociation equilibrium constant of this initial complex is 10 and 0.45 mM for DFO and L1, respectively. The first-order rate constant for its dissociation into free citrate and chelator-Fe is 0.04 and 0.02 s(-1) for DFO and L1, respectively. These results indicate that, at concentrations close to what is achieved in vivo during chelation therapy (10-100 mu M), although DFO has a thermodynamic advantage over L1, the latter acts 10 times faster than DFO in mobilizing citrate-bound iron. This large difference in the kinetics of iron removal is mainly due to the difference in the equilibrium dissociation constant of the initial complex between the chelator and Fe-citrate. The ability of a chelating agent to make a transitory complex with the iron bound to its biological carrier seems to be a major determinant in the kinetics of transfer. Our data show that the reaction pathway for the removal of Fe from citrate by DFO and L1 is different from what was observed with transferrin. Unlike transferrin, L1 (and to a lesser extent DFO) is able to directly interact with the polymeric iron-citrate; thus, depolymerization of Fe-citrate is not the rate-limiting step in the kinetics of transfer.