Hsp90 is a molecular chaperone that acts in concert with Hsp70 to mediate the folding of many important regulatory proteins (e.g., protein kinases) into functional conformations. The chaperone activity of Hsp90 is primarily regulated by its cochaperones. For example, the Hsp90 cochaperone Cdc37 recruits Hsp90 to protein kinases as well as inhibiting its ATPase activity to promote the binding of Hsp90 to protein kinases. Harc is a structurally related Hsp90 cochaperone with a three-domain structure in which the middle domain binds Hsp90. In contrast to Cdc37 though, Harc also binds to Hsp70 and Hop (Hsp70/Hsp90 organizing protein). Here we demonstrate that deletion of the C-terminal domain of Harc abolished the binding of Hsp70 and Hop and reduced the affinity of Hsp90 binding to Harc. Significantly, the C-terminal domain of Harc bound Hsp70, but it did not bind Hop or Hsp90. Size exclusion chromatography of cell lysates revealed that Hop only formed a complex with Harc in the presence of Hsp90 and Hsp70, consistent with a model in which the interaction of Hop with Harc is mediated via the binding of Hop to Harc-bound Hsp90 and Hsp70. Notably, heat shock resulted in a marked decrease in the solubility of Harc, a response that was further augmented by the deletion of the C-terminal domain of Harc. This latter finding is especially interesting given that bioinformatics analysis indicated that cells may express splice variants of Harc that encode C-terminally truncated Harc isoforms. Together, these findings indicate that the C-terminal domain of Harc is a key determinant of its cochaperone functions.