Pseudomonas aeruginosa placed across a dialysis membrane from [55Fe]transferrin caused the mobilization of the iron from the transferrin side to the bacterial or dialysate side of the membrane. Although the bacteria were capable of obtaining iron from transferrin for growth, the siderophores of P. aeruginosa failed to convert iron bound to transferrin into dialyzable, low-molecular-weight chelates. The crucial factor produced by the bacteria which was not present when the siderophores were added alone was the acid produced from the glucose minimal medium. The siderophores mobilized considerable iron from transferrin when used in the dialysis assay at pH values between 5.0 and 6.0, values which were commonly found during incubation of bacteria in the assays. When the siderophores were tested individually, pyoverdin was more effective than pyochelin in mobilizing iron across dialysis membranes at pH values of 5.0 and 6.0, but neither had appreciable activity at pH 7.4. The amounts of iron mobilized from conalbumin were comparable to the amounts from transferrin, but there was negligible release from lactoferrin at the three pH values. When the two siderophores were combined, the level of iron mobilization was identical to that demonstrated by pyoverdin alone. When the dialysis membrane was removed and the bacteria were mixed with the siderophores and transferrin, pyoverdin was again more active than pyochelin in mediating iron transport. Although no pyochelin-mediated iron mobilization could be detected at pH 7.4, there was transport. Therefore, the bacteria appeared to be aiding the siderophores in iron mobilization from transferrin.