The effect of serum and serum proteins on enterobactin- and aerobactin-mediated utilization of transferrin iron has been investigated. Serum was found to impede transfer of iron from iron transferrin to enterobactin and from [55Fe]ferric enterobactin to cells of Escherichia coli BN3040 Na 1R iuc . In contrast, serum had essentially no effect on the rate of these reactions mediated by aerobactin. Three purified serum proteins, human serum albumin, bovine serum albumin, and human immunoglobulin, were comparable to human serum in their selective ability to interfere with the transfer of 55Fe from [55Fe]ferric enterobactin to E. coli BN3040 Na 1R iuc . The inhibitory effect of human serum albumin on the enterobactin-mediated transfer of iron from [55Fe]transferrin was enhanced by preincubation of the protein with the siderophore. Pretreatment of the bacterial cells with human serum albumin did not affect the rate of utilization of siderophore iron. A linear, reciprocal relationship was found to hold for human albumin concentration vs. the first-order rate constant ( kobsd ) for the velocity of iron transfer from iron transferrin to enterobactin. Binding of serum albumin to enterobactin increased the intensity of the near-ultraviolet absorption band of the siderophore and shifted it to longer wavelengths. The stoichiometry of binding to human and bovine serum albumins was established as 1:1, and the binding constant for both enterobactin and ferric enterobactin was estimated to be in the range 1 X 10(4)-1.2 X 10(5) M-1. These results indicate that serum albumin may act synergistically with other factors in the serum, such as transferrin, to limit iron supply and in this way restrict the growth of invading microorganisms.