MAS17 (MAS22) is an essential component of the import receptor complex in the yeast mitochondrial outer membrane. MAS17 consists of three distinct domains: the N-terminal cytosolic domain, the internal membrane-spanning domain, and the C-terminal intermembrane space domain. In the present study, we examined the roles of the C-terminal domain of MAS17, which is rich in acidic amino acids, in protein import into mitochondria both in vivo and in vitro. Cells expressing MAS17 delta 120-152, a mutant MAS17 lacking the C-terminal acidic domain, could grow as fast as those expressing wild-type MAS17, while cells expressing MAS17 delta 97-152, a mutant MAS17 lacking both the intermembrane space and the membrane-spanning domains, stopped growing as soon as wild-type MAS17 was depleted. MAS17 delta 120-152 was correctly integrated into the mitochondrial outer membrane like wild-type MAS17. Mitochondria containing MAS17 delta 120-152 instead of wild-type MAS17 could import both authentic and artificial mitochondrial precursor proteins nearly as efficiently as wild-type mitochondria in vitro. These results suggest that the C-terminal intermembrane space domain of MAS17 is not essential for targeting or functions of MAS17.