Mitophagy is a crucial process that preserves normal cell function in mammals by selective removal of defective mitochondria. In dissecting the molecular pathways that mediate its efficiency we have now identified an important role for the mitochondrial Translocator Protein (mTSPO), an 18-kDa protein localized on the outer mitochondrial membrane and over-expressed in pathologies such as brain inflammation and cancers (Gatliff and Campanella, 2012). In human, murine and canine cell lines, mTSPO was transiently over-expressed or silenced via mTSPO cDNA (+mTSPO) or siRNA (-mTSPO) using Lipofectamine and Ca2+ Phosphate transfection techniques. An empty vector (C) or non-silencing siRNA was used in control cells. Cells were harvested 36 h post transfection with modulation confirmed via western blot analysis (n>5). Fluorescence (F) or luminescence-based assays were employed to monitor alterations in core aspects of mitochondrial physiology (Seneviratne et al, 2012). Values are means ± S.E.M., compared by ANOVA. +mTSPO led to reduced mitochondrial Ca2+ uptake after challenge with an InsP3-generating stimulus, ATP (100µM) (maximum Rhod-2 F (arbitrary units (A.U.): C 0.51±0.02, +mTSPO 0.29±0.03, -mTSPO 0.76±0.03; p<0.001; n>10); lower mitochondrial membrane potential (ΔΨm) (tetramethylrhodamine methyl ester (TMRM) F (A.U.): C 1.00±0.1 +mTSPO 0.84±0.09 -mTSPO 1.93±0.17; p<0.001; n>15); increased reactive oxygen species (ROS) production (rate of increase of oxidized dihydroethidium F: C 2.37±0.19 +mTSPO 4.7±0.34 -mTSPO 0.42±0.05; p<0.001; n>10) and limited ATP generation. The opposite was observed across parameters in -mTSPO cells. To examine if these effects were due to changes in mitophagy, 20 μM carbonylcyanide-4-trifluoromethoxyphenylhydrazone (FCCP), an uncoupler, was applied for 2 h to stimulate mitophagy in cells transiently co-transfected with light-chain III-green fluorescent protein (LC3-GFP), an autophagosome (AP) marker, and mitochondria-targeted red fluorescent protein (mtRFP). Co-localization of the signals was quantified using Volocity software. Mitochondrial ubiquitination -pivotal in removal of defective mitochondria via the PINK1/Parkin pathway [Narendra et al, 2010]- was explored by immunolabeling in transfected cells and confirmed via immunoblotting of mitochondrial fractions (n=2). In summary +mTSPO cells had limited mitochondria containing APs (C 0.404±0.034 +mTSPO 0.102±0.024 -mTSPO 0.586±0.065; n>20; p<0.05) by preventing ubiquitination of mitochondrial proteins (% cells with mitochondrial ubiquitination: C 25.4±3.2 +mTSPO 4.9±2.4 -mTSPO 53.4±3.7 p<0.05 n>3) while in -mTSPO cells basal and activated mitophagy were augmented. These data suggest mTSPO may limit mitochondrial quality by restricting the efficiency of mitophagy. Where mTSPO levels surpass normal, unphysiological accumulation of defective mitochondria may cause and sustain cellular and tissue degeneration.