Newly synthesized mitochondrial precursor proteins have to become unfolded to cross the mitochondrial membranes. This unfolding is achieved primarily by mitochondrial Hsp70 (mtHsp70) for presequence-containing precursor proteins. However, the membrane potential across the inner membrane (ΔΨ) could also contribute to unfolding of short-presequence containing mitochondrial precursor proteins. Here we investigated the role of ΔΨ in mitochondrial protein unfolding and import. We found that the effects of mutations in the presequence on import rates are correlated well with the hydrophobicity or ability to interact with import motor components including mtHsp70, but not with ΔΨ (negative inside). A spontaneously unfolded precursor protein with a short presequence is therefore trapped by motor components including mtHsp70, but not ΔΨ, which could cause global unfolding of the precursor protein. Instead, ΔΨ may contribute the precursor unfolding by holding the presequence at the inner membrane for trapping of the unfolded species by the import motor system.