Heat shock proteins 90 (Hsp90) are chaperones that promote the proper folding of other proteins under high temperature stress situations. Hsp90s are highly conserved and ubiquitous proteins, and in mammalian cells, they are localized in the cytoplasm, endoplasmic reticulum, and mitochondria. Cytoplasmic Hsp90 are named Hsp90α and Hsp90β and differ mainly in their expression pattern: Hsp90α is expressed under stress conditions, while Hsp90β is a constitutive protein. Structurally, both share the same characteristics by presenting three well-conserved domains, one of which, the N-terminal domain, has a binding site for ATP to which various drugs targeting this protein, including radicicol, can bind. The protein is mainly found in dimeric form and adopts different conformations depending on the presence of ligands, co-chaperones and client proteins. In this study, some aspects of structure and thermal unfolding of cytoplasmic human Hsp90 were analysed by infrared spectroscopy. The effect on Hsp90β of binding with a non-hydrolysable ATP analogue and radicicol was also examined. The results obtained showed that despite the high similarity in secondary structure the two isoforms exhibit substantial differences in their behaviour during thermal unfolding, as Hsp90α exhibits higher thermal stability, slower denaturation process and different event sequence during unfolding. Ligand binding strongly stabilizes Hsp90β and slightly modifies the secondary structure of the protein as well. Most likely, these structural and thermostability characteristics are closely related to the conformational cycling of the chaperone and its propensity to exist in monomer or dimer form.