Requirement of the mitochondrial import receptor Tom20 in protein import into mammalian mitochondria was studied in vitro and in cultured cells. Import of human and rat pre-ornithine transcarbamylase (pOTC), pig pre-aspartate aminotransferase (pAAT) and rat serine: pyruvate aminotransferase (pSPT) was inhibited by delta hTom20 that lacks the NH2-terminal transmembrane domain of human Tom20 (hTom20). Import of these preproteins was also inhibited by anti-Tom20. The inhibitions by delta hTom20 and anti-hTom20 were the strongest for human pOTC, followed by rat pOTC, pAAT and pSPT. Coexpression of human pOTC and hTom20 in COS-7 cells followed by immunoblot analysis showed that overexpression of hTom20, but not delta hTom20, decreases production of mature OTC. In pulse-chase experiments, pOTC was synthesized and rapidly processed to the mature form. Coexpression of hTom20, but not delta hTom20, resulted in a decrease of pOTC processing, probably due to an imbalance of the normal stoichiometry of the receptor complex. These results show that both in vitro and in intact cells, Tom20 is involved in mitochondrial protein import in higher animals and that the requirement for Tom20 is different for different preproteins.