The role of ATP in the matrix for the import of precursor proteins into the various mitochondrial subcompartments was investigated by studying protein translocation at experimentally defined ATP levels. Proteins targeted to the matrix were neither imported or processed when matrix ATP was depleted. Import and processing of precytochrome b2 (pb2), a precursor carrying a bipartite presequence, into the intermembrane space was also strongly dependent on matrix ATP. Preproteins, consisting of 220 or more residues of pb2 fused to dihydrofolate reductase, showed the same requirement for matrix ATP, whereas the import of shorter fusion proteins (up to 167 residues of pb2) was largely independent of matrix ATP. For those intermembrane-space-targeted proteins that did need matrix ATP, the dependence could be relieved either by unfolding these proteins prior to import or by introducing a deletion into the mature portion of the protein thereby impairing the tight folding of the cytochrome b2 domain. These results suggest the following: (a) The import of matrix-targeted preproteins, in addition to a membrane potential delta psi, requires matrix ATP [most likely to facilitate reversible binding of mitochondrial heat-shock protein 70 (mt-Hsp70) to incoming precursors], for two steps, securing the presequence on the matrix side of the inner membrane and for the completion of translocation; (b) in the case of intermembrane-space-targeted precursors with bipartite signals, the function of ATP/mt-Hsp70 is not obligatory, as components of the intermembrane-space-sorting pathway may substitute for ATP/mt-Hsp70 function (however, if a tightly folded domain is present in the precursor, ATP/mt-Hsp70 is indispensable); (c) unfolding on the mitochondrial surface of tightly folded segments of preproteins is facilitated by matrix-ATP/mt-Hsp70.