Systemic anthrax infections can be characterized as proceeding in stages, beginning with an early intracellular establishment stage within phagocytes that is followed by extracelluar stages involving massive bacteraemia, sepsis and death. Because most bacteria require iron, and the host limits iron availability through homeostatic mechanisms, we hypothesized that B. anthracis requires a high-affinity mechanism of iron acquisition during its growth stages. Two putative types of siderophore synthesis operons, named Bacillus anthracis catechol, bac (anthrabactin), and anthrax siderophore biosynthesis, asb (anthrachelin), were identified. Directed gene deletions in both anthrabactin and anthrachelin pathways were generated in a B. anthracis (Sterne) 34F2 background resulting in mutations in asbA and bacCEBF. A decrease in siderophore production was observed during iron-depleted growth in both the DeltaasbA and DeltabacCEBF strains, but only the DeltaasbA strain was attenuated for growth under these conditions. In addition, the DeltaasbA strain was severely attenuated both for growth in macrophages (MPhi) and for virulence in mice. In contrast, the DeltabacCEBF strain did not differ phenotypically from the parental strain. These findings support a requirement for anthrachelin but not anthrabactin in iron assimilation during the intracellular stage of anthrax.