Steroidogenic acute regulatory protein (StAR) is a mitochondrial protein essential for massive synthesis of steroid hormones in the adrenal and the gonads. Our studies suggest that once synthesized on free polyribosomes, StAR preprotein either associates with the outer mitochondrial membrane to mediate transfer of cholesterol substrate required for steroidgenesis, or it is degraded by the proteasome. Proteasome inhibitors can prevent the turnover of StAR preprotein and other matrix-targeted preproteins. Once imported, excessive accumulation of inactive StAR in the matrix is avoided by a rapid turnover. Unexpectedly, mitochondrial StAR turnover can be inhibited by two proteasome inhibitors, i.e., MG132 and clasto-lactacystin beta-lactone, but not epoxomicin. Use of those inhibitors and immuno-electron microscopy data enabled a clear distinction between two pools of intra-mitochondrial StAR, one degraded by matrix protease(s) shortly after import, while the rest of the protein undergoes a slower and inhibitor resistant degradation following translocation onto to the matrix face of the inner membranes.