Hsp90 is a ubiquitous, well-conserved molecular chaperone involved in the folding and stabilization of diverse proteins. Beyond its capacity for general protein folding, Hsp90 influences a wide array of cellular signaling pathways that underlie key biological and disease processes. It has been proposed that Hsp90 functions as a molecular clamp, dimerizing through its carboxy-terminal domain and utilizing ATP binding and hydrolysis to drive large conformational changes including transient dimerization of the amino-terminal and middle domains. We have determined the 2.6 A X-ray crystal structure of the carboxy-terminal domain of htpG, the Escherichia coli Hsp90. This structure reveals a novel fold and that dimerization is dependent upon the formation of a four-helix bundle. Remarkably, proximal to the helical dimerization motif, each monomer projects a short helix into solvent. The location, flexibility, and amphipathic character of this helix suggests that it may play a role in substrate binding and hence chaperone activity.