Complex formation of the Hsp70 chaperone DnaK with the fluorescence-labeled peptide ALLLSAPRR shows a very rapid first phase that has as yet not been observed with other peptides. This first phase is completed within the dead time (1-2 ms) of the stopped-flow instrument and corresponds to two thirds of the total increase in fluorescence. It occurs both in the presence and in the absence of ATP and is followed by a fast, a slow and a very slow step. These binding kinetics that are vastly different from those observed with other peptides might indicate the existence of a second substrate-binding site of DnaK.