The molecular chaperone heat shock protein 90 (Hsp90) is required for the folding and activation of numerous essential signaling proteins. Hsp90 is generally thought to transition between an open (apo) and a closed (ATP) conformation in response to nucleotide. Here, 3D single-particle reconstructions of Escherichia coli and yeast Hsp90 homologs establish the existence of two distinct nucleotide-stabilized conformations (ATP, ADP) in addition to an apo extended state, supporting previous structural work. However, single-particle matching methods reveal that, rather than being irreversibly determined by nucleotide, a species-dependent dynamic conformational equilibrium exists between states. Using crosslinking methods, we trap transient nucleotide-specific states of yeast and human Hsp90 and establish that the apo, ATP, and ADP states are universal. These data support a conserved three-state chaperone cycle where the conformational equilibrium varies between species, implicating evolutionary tuning to meet the particular client protein and metabolic environment of an organism.