The role of nucleoside triphosphates (NTPs) in the import of porin into the mitochondrial outer membrane was investigated with two forms of the porin precursor: the in vitro synthesized biosynthetic precursor (bs- porin) and a water-soluble form of porin (ws-porin) obtained by subjecting the membrane-derived porin to an acid-base treatment (exposure to trichloroacetic acid, followed by alkali and rapid neutralization). The import of ws-porin into mitochondria did not require NTPs, whereas the import of bs-porin required NTPs. In other characteristics, such as binding to a specific receptor protein on the mitochondrial surface, two-step insertion into the outer membrane, and formation of specific membrane channels, ws-porin was indistinguishable from bs-porin. Thus, the acid-base treatment applied in the preparation of ws-porin can substitute for the NTP-requiring step in mitochondrial protein import. We conclude that NTPs are required for unfolding mitochondrial precursor proteins ("translocation competent folding").