Interaction of preproteins with the heat shock protein Hsp70 in the mitochondrial matrix is required for driving protein transport across the mitochondrial inner membrane. Binding of mt-Hsp70 to the protein Mim44 of the inner membrane import site seems to be an essential part of an ATP-dependent reaction cycle. However, the available results on the role played by ATP are controversial. Here we demonstrate that the mt-Hsp70.Mim44 complex contains ADP and that a nonhydrolyzable analog of ATP dissociates the mt-Hsp70.Mim44 complex in the presence of potassium ions. The previously reported requirement of ATP hydrolysis for complex dissociation was due to the use of a nonphysiological concentration of sodium ions. In the presence of potassium ions, mt-Hsp70 undergoes a conformational change that is not observed with a mutant Hsp70 defective in binding to Mim44. The mutant Hsp70 is able to bind substrate proteins, differentiating binding to Mim44 from binding to substrate proteins. We conclude that binding of ATP, not hydrolysis, is required to dissociate the mt-Hsp70.Mim44 complex and that the reaction cycle includes an ATP-induced conformational change of mt-Hsp70.