ATPase and peptide-binding activity of recombinant human heat shock proteins HSP70A1B and HSC70 and two hybrid proteins derived from them was investigated. UV-spectral recorded data were used to characterize conformational rearrangements induced by domain replacement or HSP70-peptide interaction. It was shown that the N-terminal domain dramatically affects the substrate specificity of the C-terminal peptidebinding domain, which puts forward a new hypothesis for HSP70 chaperone machinery. On the other hand, the peptide-binding domain affected the ATPase activity of the recombinant proteins. There was a linear relationship between the ATPase activity and the peptide complex percentage. This connection can be used for quantification of HSP70 complexes with unlabeled peptides.