Nucleotide dependent conformational changes of the constitutively dimeric molecular chaperone Hsp90 are integral to its molecular mechanism. Recent full-length crystal structures of Hsp90 homologs reveal large-scale quaternary domain rearrangements upon the addition of nucleotides. While previous work has shown the importance of C-terminal domain dimerization for efficient ATP hydrolysis, which should imply cooperativity, other studies suggest that the two ATPases function independently. Using the crystal structures as a guide, we examined the role of intra- and inter-monomer interactions in stabilizing the ATPase activity of a single active site within an intact dimer. This was accomplished by creating heterodimers that allow us to differentially mutate each monomer probing the context in which particular residues are important for ATP hydrolysis. While the ATPase activity of each monomer can function independently, we found that the activity of one monomer could be inhibited by the mutation of hydrophobic residues on the trans N-terminal domain (opposite monomer). Furthermore, these trans interactions are synergistically mediated by a loop on the cis middle domain. This loop contains hydrophobic residues as well as a critical arginine that provides a direct linkage to the -phosphate of bound ATP. Small-angle x-ray scattering demonstrates that deleterious mutations block domain closure in the presence of AMPPNP, providing a direct linkage between structural changes and functional consequences. Together these data indicate that both the cis- and trans-monomer and intra- and inter-domain interactions cooperatively stabilize the active conformation of each active site and help explain the importance of dimer formation.