The steroidogenic acute regulatory protein (StAR) facilitates the movement of cholesterol into mitochondria to initiate steroidogenesis, but its site of action on the mitochondria has been uncertain. One model states that StAR has a fairly rigid structure and functions in the intramembranous space (IMS) where it transports cholesterol from the outer mitochondrial membrane (OMM) to the inner mitochondrial membrane (IMM); another model states that StAR works solely on or in the OMM and undergoes a partially open molten globule conformation while picking up and discharging cholesterol. We designed, built and tested a series of StAR fusion proteins designed to immobilize StAR on the OMM, the IMS, or the matrix side of the IMM. Only the constructs at the OMM were active, either in vivo or in vitro. As these data indicated that StAR acts at or in the OMM we hypothesized that StAR' s activity would be proportional to the amount of time it spends on the OMM. To test this hypothesis, we built a series of StAR proteins with altered mitochondrial leaders designed to speed or slow StAR's mitochondrial entry. Cell transfections showed that the constructs that slowed entry had more activity and those designed to speed entry had less activity. Analysis of import kinetics in vitro confirmed that these constructs accelerated import inversely proportional to their activity. These data show that StAR works only on the OMM, providing an unusual example of a protein that exerts its biological activity in a cellular location it occupies only transiently, rather than in the location (the matrix) to which it is targeted.