In the matrix of mitochondria, protein homeostasis is maintained by molecular chaperones and proteases very similar to those found in the cytosol of bacteria. This network of proteins ensures mitochondrial protein homeostasis under normal cellular conditions, however some members also play crucial roles under conditions of protein folding stress. Matrix proteases, at least in Caenorhabditis elegans, appear to play a role in both the clearance of non-native proteins and the activation of the mitochondrial unfolded protein response (mtUPR). Specifically, the matrix AAA+ protease CLPXP is proposed to initiate mtUPR through the degradation of unfolded proteins, resulting in the generation of activating peptides. However, little is known about the role of matrix proteases in the signalling pathway of mtUPR in mammalian cells. To better understand the molecular details of protein homeostasis including mtUPR activation in mammals, we have employed import-chase assays using isolated mammalian mitochondria, to determine which matrix protease (i.e. CLPXP or LONM) is responsible for the clearance of unfolded aggregation prone proteins. Furthermore, degradation of unfolded model and authentic mitochondrial matrix proteins has been examined in vitro using purified recombinant CLPXP and LONM. We will describe the outcomes of both the in organello and in vitro studies reporting on the division of labour between LONM and CLPXP in the mitochondrial matrix and the implications for signalling of mtUPR in mammals.