The ability of malleobactin to mobilize iron from transferrin and lactoferrin was examined in an equilibrium dialysis assay in the absence of bacteria. Malleobactin was capable of removing iron from both transferrin and lactoferrin at pH values of 7.4, 6.0, and 5.0. However, the levels of iron mobilization were greater for transferrin than for lactoferrin at all the pH values used in the assay. The ability of Pseudomonas pseudomallei to acquire iron from 30% iron-saturated transferrin and K562 human erythroleukemic cells was compared in parallel cultures as described previously (J. H. Brock, P. H. Williams, J. Liceaga, and K. G. Woldridge, Infect. Immun. 59:3185-3190, 1991). P. pseudomallei U7 tended to acquire iron from transferrin. In contrast, P. aeruginosa PAO and P. cepacia Pc275C acquired iron from both sources. P. cepacia H1721, which does not produce detectable siderophores, but can utilize malleobactin, pyochelin, and azurechelin as iron sources, was used in a similar experiment. Addition of malleobactin resulted in iron uptake only from transferrin, whereas pyochelin and azurechelin promoted iron uptake from both sources. When the siderophores were incubated with K562 cells alone, malleobactin was less efficient at removing iron from cells than pyochelin and azurechelin. It was also determined that malleobactin was less effective in binding to or entering cells than pyochelin and azurechelin. These results suggest that malleobactin can acquire iron more effectively from host proteins than from cellular sources. Pyochelin and azurechelin can acquire cell-derived iron in addition to iron bound to host proteins.