Biotin synthase (BioB) is an iron-sulfur enzyme that catalyzes the last step in biotin biosynthesis, the insertion of sulfur between the C6 and C9 atoms of dethiobiotin to complete the thiophane ring of biotin. Recent in vitro experiments suggest that the sulfur is derived from a [2Fe-2S](2+) cluster within BioB, and that the remnants of this cluster dissociate from the enzyme following each turnover. For BioB to catalyze multiple rounds of biotin synthesis, the [2Fe-2S](2+) cluster in BioB must be reassembled, a process that could be conducted in vivo by the ISC or SUF iron-sulfur cluster assembly systems. The bacterial ISC system includes HscA, an Hsp70 class molecular chaperone, whose yeast homologue has been shown to play an important but nonessential role in assembly of mitochondrial FeS clusters in Saccharomyces cerevisiae. In this work, we show that in Escherichia coli, HscA significantly improves the efficiency of the in vivo assembly of the [2Fe-2S](2+) cluster on BioB under conditions of low to moderate iron. In vitro, we show that HscA binds with increased affinity to BioB missing one or both FeS clusters, with a maximum of two HscA molecules per BioB dimer. BioB binds to HscA in an ATP/ADP-independent manner, and a high-affinity complex is also formed with a truncated form of HscA that lacks the nucleotide binding domain. Further, the BioB-HscA complex binds the FeS cluster scaffold protein IscU in a noncompetitive manner, generating a complex that contains all three proteins. We propose that HscA plays a role in facilitating the transfer of FeS clusters from IscU into the appropriate target apoproteins such as biotin synthase, perhaps by enhancing or prolonging the requisite protein-protein interaction.