Aspergillus fumigatus is an opportunistic fungal pathogen that causes life-threatening infections in immunocompromised patients. Despite low levels of free iron, A. fumigatus grows in the presence of human serum in part because it produces high concentrations of siderophores. The most abundant siderophores produced by A. fumigatus are N',N'',N'''-triacetylfusarinine C (TAF) and ferricrocin, both of which have thermodynamic iron binding constants that theoretically allow them to remove transferrin (Tf)-bound iron. Urea-polyacrylamide gel electrophoresis was used to measure the change in concentration of Tf species incubated with TAF or ferricrocin. The rate of removal of iron from diferric Tf by both siderophores was measured, as were the individual microscopic rates of iron removal from each Tf species (diferric Tf, N-terminal monoferric Tf and C-terminal monoferric Tf). TAF removed iron from all Tf species at a faster rate than ferricrocin. Both siderophores showed a preference for removing C-terminal iron, evidenced by the fact that k(1C) and k(2C) were much larger than k(1N) and k(2N). Cooperativity in iron binding was observed with TAF, as the C-terminal iron was removed by TAF much faster from monoferric than from diferric Tf. With both siderophores, C-terminal monoferric Tf concentrations remained below measurable levels during incubations. This indicates that k(2C) and k(1C) are much larger than k(1N). TAF and ferricrocin both removed Tf-bound iron with second-order rate constants that were comparable to those of the siderophores of several bacterial pathogens, indicating they may play a role in iron uptake in vivo and thereby contribute to the virulence of A. fumigatus.