Mitochondrial uncoupling protein 2 (UCP2) is implicated in a wide range of pathophysiological processes, including immunity and diabetes mellitus, but its rapid degradation remains uncharacterized. Using pharmacological proteasome inhibitors, immunoprecipitation, dominant negative ubiquitin mutants, [corrected] cellular fractionation and siRNA techniques, we demonstrate the involvement of the ubiquitin-proteasome system in the rapid degradation of UCP2. Importantly, we resolve the issue of whether intramitochondrial proteins can be degraded by the cytosolic proteasome by reconstituting a cell-free system that shows rapid proteasome-inhibitor-sensitive UCP2 degradation in isolated, energised mitochondria presented with an ATP regenerating system, ubiquitin and 26S proteasome fractions. These observations provide the first demonstration that a mitochondrial inner membrane protein is degraded by the cytosolic ubiquitin-proteasome system.