During protein import into mitochondria, matrix-localized mitochondrial hsp70 (mhsp70) interacts with the inner membrane protein Tim44 to pull a precursor across the inner membrane. We have proposed that the Tim44-mhsp70 complex functions as an ATP-dependent Xtranslocation motorX that exerts an inward force on the precursor chain. To clarify the role of ATP in mhsp70-driven translocation, we tested the effect of the purified ATP analogues AMP-PNP and ATP gamma S on the Tim44-mhsp70 interaction. Both analogues mimicked ATP by causing dissociation of mhsp70 from Tim44. ADP did not disrupt the Tim44-mhsp70 complex, but did block the ATP-induced dissociation of this complex. In the presence of ADP, mhsp70 can bind simultaneously to Tim44 and to a peptide substrate. These data are consistent with a model in which mhsp70 first hydrolyzes ATP, then associates tightly with Tim44 and a precursor protein, and finally undergoes a conformational change to drive translocation.