SUMMARY Mps1 protein kinase is required for accurate chromosome segregation during the mitotic checkpoint. The molecular chaperone Heat Shock Protein 90 (Hsp90) has been linked to Mps1 activity, however the molecular mechanismsunderlying this regulatory process remain elusive. We report that Mps1 directly phosphorylates a conserved threonine residue (T101 in yeast Hsp90 and T115 in human Hsp90) in the N domain of Hsp90. This phosphorylation regulates chaperone function by reducingHsp90 ATPase activity andfosteringits association with kinase client proteins including Mps1. Phosphorylation of T101is also essential for the mitotic checkpoint because it confers Mps1 stability and activity.Additionally,Mps1 phosphorylation of Hsp90 sensitizes cells to Hsp90 inhibitors and elevated Mps1 levels confer tumor selectivity on Hsp90 drugs.Finally,we identified Cdc14 as the phosphatasethat dephosphorylatesT101 and disruptsthe Mps1-Hsp90 interaction. This leads to degradation of Mps1,thusproviding a mechanism for its inactivation and mitotic exit.