Hsp90, an abundant heat shock protein that is highly expressed even under physiological conditions, is involved in the folding of key molecules of the cellular signal transduction system such as kinases and steroid receptors. It seems to contain two chaperone sites differing in substrate specificity. Binding of ATP or the antitumor drug geldanamycin alters the substrate affinity of the N-terminal chaperone site, whereas both substances show no influence on the C-terminal one. In wild-type Hsp90 the fragments containing the chaperone sites are connected by a highly charged linker of various lengths in different organisms. As this linker region represents the most striking difference between bacterial and eukaryotic Hsp90s, it may be involved in a gain of function of eukaryotic Hsp90s. Here, we have analyzed a fragment of yeast Hsp90 consisting of the N-terminal domain and the charged region (N272) in comparison with the isolated N-terminal domain (N210). We show that the charged region causes an increase in the affinity of the N-terminal domain for nonnative protein and establishes a crosstalk between peptide and ATP binding. Thus, the binding of peptide to N272 decreases its affinity for ATP and geldanamycin, whereas the ATP-binding properties of the monomeric N-terminal domain N210 are not influenced by peptide binding. We propose that the charged region connecting the two chaperone domains plays an important role in regulating chaperone function of Hsp90.