Steroidogenic acute regulatory protein (StAR) plays a critical role in steroid hormone biosynthesis, presumably by facilitating the delivery of cholesterol to P450scc in the inner mitochondrial membranes. StAR is synthesized as a 37-kDa preprotein that is processed to a 30-kDa mature form by cleavage of an N-terminal mitochondrial import sequence. To identify structural features required for StAR biological activity, we mutated the human StAR cDNA, including the deletion of N- and C-terminal sequences, and examined the ability of the mutants to promote steroidogenesis and enter the mitochondria of transfected COS-1 cells. Deletion of up to 62 residues from the N terminus (N-62) did not significantly affect steroidogenesis-enhancing activity. The N-terminal deletion mutants were associated with mitochondria-enriched fractions, but import and processing were progressively impaired with increasing length of the deletion. Immunogold electron microscopy and in vitro import assays showed that the active N-62 mutant was not imported into the mitochondria. Removal of the 28 C-terminal amino acids (C-28) inactivated StAR. Deletion of the C-terminal 10 amino acids (C-10) reduced steroidogenic activity by 53%, while truncation of the last 4 amino acids had no effect. The C-28 mutant StAR was not efficiently imported into mitochondria or processed, whereas some of the C-10 mutant was processed, indicating that import had occurred. We conclude that in the COS-1 cell system used, StAR does not need to enter into mitochondria to stimulate steroidogenesis and that residues in the C terminus are essential for steroidogenesis-enhancing activity. These findings imply that StAR acts via C-terminal domains on the outside of the mitochondria.