During growth under iron limitation, Bacillus cereus and Bacillus anthracis, two human pathogens from the Bacillus cereus group of Gram-positive bacteria, secrete two siderophores, bacillibactin (BB) and petrobactin (PB), for iron acquisition via membrane-associated substrate-binding proteins (SBPs) and other ABC transporter components. Since PB is associated with virulence traits in B. anthracis, the PB-mediated iron uptake system presents a potential target for antimicrobial therapies; its characterization in B. cereus is described here. Separate transporters for BB, PB, and several xenosiderophores are suggested by (55)Fe-siderophore uptake studies. The PB precursor, 3,4-dihydroxybenzoic acid (3,4-DHB), and the photoproduct of FePB (FePB(nu)) also mediate iron delivery into iron-deprived cells. Putative SBPs were recombinantly expressed, and their ligand specificity and binding affinity were assessed using fluorescence spectroscopy. The noncovalent complexes of the SBPs with their respective siderophores were characterized using ESI-MS. The differences between solution phase behavior and gas phase measurements are indicative of noncovalent interactions between the siderophores and the binding sites of their respective SBPs. These studies combined with bioinformatics sequence comparison identify SBPs from five putative transporters specific for BB and enterobactin (FeuA), 3,4-DHB and PB (FatB), PB (FpuA), schizokinen (YfiY), and desferrioxamine and ferrichrome (YxeB). The two PB receptors show different substrate ranges: FatB has the highest affinity for ferric 3,4-DHB, iron-free PB, FePB, and FePB(nu), whereas FpuA is specific to only apo- and ferric PB. The biochemical characterization of these SBPs provides the first identification of the transporter candidates that most likely play a role in the B. cereus group pathogenicity.