Hsp90 is one of the most abundant proteins in the cytosol of eukaryotic cells. Under physiological conditions Hsp90 has been shown to play a major role in several specific signaling pathways, including maturation of various kinases and maintenance of steroid receptors in an activable state. It is well established that the level of Hsp90 increases severalfold under stress conditions, and it has been shown that the chaperone function of Hsp90 is ATP-independent. Although yeast Hsp90 does not bind ATP, as determined by a number of methods monitoring tight binding, ATP-dependent functions of Hsp90 in the presence of co-factors and elevated temperatures are still under discussion.Here, we have reinvestigated ATP-binding properties and ATPase activity of human Hsp90 under various conditions. We show that human Hsp90 does not bind ATP tightly and does not exhibit detectable ATPase activity. However, using electron spin resonance spectroscopy, weak binding of spin-labeled ATP analogues with halfmaximal binding at 400 M ATP was detected. The functional significance of this weak interaction remains enigmatic.Under stress conditions, e.g. high temperatures, cells overexpress a distinct set of proteins, the so-called heat shock or stress proteins. The major classes of heat shock proteins, Hsp90, 1 Hsp70, Hsp60, and small Hsps, are thought to function as molecular chaperones during protein folding (1-3). The mechanisms of these chaperone functions are still under intensive investigation. In the case of Hsp70 and Hsp60, ATP binding and hydrolysis is a major requirement in chaperone-mediated folding (1). In vivo experiments suggest that Hsp90, one of the most abundant and conserved heat shock proteins, is a specific chaperone involved in regulating signal transduction pathways by assisting structural changes of certain kinases and steroid receptors (4 -8). In addition, results from in vitro studies highlight the general chaperone activities of Hsp90 (2, 3). Like other chaperones, Hsp90 performs at least part of its activity in association with specific partner proteins (9 -14), some of which seem to function as molecular chaperones themselves (15,16). In this context, the involvement of ATP in Hsp90 function is still a controversial subject (11)(12)(13)(17)(18)(19), and ATP binding as well as ATPase activity of Hsp90 have been reported previously (18, 20 -23). In contrast to these findings, we have shown that yeast Hsp90 does not bind ATP (17) by means of assays that are reflecting structural changes of the observed protein in the presence of ligand, detecting binding of the protein to immobilized ATP or binding of fluorescencelabeled ATP analogues to Hsp90. These observation enabled only the investigation of tight interactions between ATP and Hsp90. New results concerning p23, a partner protein of Hsp90 that is thought to play an important role in Hsp90/steroid receptor complexes, readdressed the possibility that human Hsp90 is an ATP-binding protein (18). This brought up the question of whether human Hsp90 differs in its ATP...