The Steroidogenic Acute Regulatory (StAR) protein is a mitochondrial protein required for the transport of cholesterol substrate to the P450scc enzyme located in the inner mitochondrial membranes of steroid producing cells. This study suggests that the acute regulation of the rodent StAR gene in the ovary is mediated by two factors, C/EBPbeta and GATA-4. Once translated, the StAR precursor protein is either imported into the mitochondria, or it is rapidly degraded in the cytosol. We predicted that in order to perpetuate StAR activity cycles, imported StAR should turn over rapidly to avoid a potentially harmful accumulation of the protein in sub-mitochondrial compartments. Pulse-chase experiments in metabolically labeled cells showed that: (a) the turnover rate of mature mitochondrial StAR protein (30kDa) is much faster (t(1/2)=4-5h) than that of other mitochondrial proteins; (b) dissipation of the inner membrane, potential (-Deltapsi) by carbonyl cyanide m-chlorophenylhydrazone (mCCCP) accelerates the mitochondrial degradation of StAR; (c) unexpectedly, the mitochondrial degradation of StAR is inhibited by MG132 and lactacystin, but not by epoxomicin. Furthermore, StAR degradation becomes inhibitor-resistant two hours after import. Therefore, these studies suggest a bi-phasic route of StAR turnover in the mitochondria. Shortly after import, StAR is degraded by inhibitor-sensitive protease(s) (phase I), whereas at later times, StAR turnover proceeds to completion through an MG132-resistant proteolytic activity (phase II). Collectively, this study defines StAR as a unique protein that can authentically be used to probe multiple proteolytic activities in mammalian mitochondria.