1,1'-Bis(4-anilino-5-naphthalenesulfonic acid) (bis-ANS), a hydrophobic fluorescent molecular probe which has been shown to bind to compact intermediate states of proteins (molten globules) and also to many nucleotide binding sites, induces a conformational change in DnaK by preferentially binding to its partially folded intermediate state (I) and thus shifting the equilibrium from favoring the native state (N) to favoring the I state. The conformational change was detected by CD, fluorescence emission, size exclusion chromatography, and small-angle X-ray scattering. The presence of bis-ANS significantly decreases the midpoint, Tm, of the initial transition (N-->I) in the thermal unfolding of DnaK, resulting in the apparent destabilization of the native state of DnaK. There is a linear correlation between the apparent free energy (reflected by Tm) of this transition and the concentration of bis-ANS. Bis-ANS does not affect the midpoint of the transition for DnaK from the intermediate to the unfolded state (U). An additional small transition from I to I*, a more expanded intermediate state, was observed, suggesting that the thermal denaturation of DnaK proceeds via a four-state (N-->I-->I*-->U) unfolding process. The addition of nucleotides, ADP or ATP, to the DnaK-bis-ANS complex causes a decrease in bis-ANS fluorescence emission due to the release of bound bis-ANS from the intermediate state of DnaK. This is due to preferential binding of the nucleotide to the native state of DnaK, resulting in a shift in the equilibrium from the intermediate toward the native state rather than the direct displacement of bis-ANS bound in the nucleotide binding site. Denaturation of DnaK induced by bis-ANS can be minimized by working at a temperature much lower than the Tm of the protein, at low dye concentration, and in the presence of nucleotide. Under these conditions, bis-ANS binds to the native state of DnaK.