Ubiquitinated endosomal proteins that are deposited into the lumens of multivesicular bodies are either sorted for lysosomal-mediated degradation or secreted as exosomes into the extracellular milieu. The mechanisms that underlie the sorting of cellular cargo proteins are currently unknown. In this study, we show that the COP9 signalosome (CSN)-associated protein CSN5 quantitatively regulated proteins that were sorted into exosomes. Western blot analysis of exosomal proteins indicated that small interfering (si)RNA knockdown of CSN5 results in increased levels of both ubiquitinated and non-ubiquitinated exo- somal proteins, including heat shock protein 70, in comparison with exosomes isolated from the super- natants of 293 cells transfected with scrambled siRNA. Furthermore, 293 cells transfected with JAB1/MPN/ Mov34 metalloenzyme domain-deleted CSN5 produced exosomes with higher levels of ubiquitinated heat shock protein 70, which did not affect non-ubiq- uitinated heat shock protein 70 levels. The loss of COP9-associated deubiquitin activity of CSN5 also led to the enhancement of HIV Gag that was sorted into exosomes as well as the promotion of HIV-1 release, suggesting that COP9-associated CSN5 regulates the sorting of a number of exosomal proteins in both a CSN5 JAB1/MPN/Mov34 metalloenzyme domain-dependent and -independent manner. We propose that COP9-associated CSN5 regulates exosomal protein sorting in both a deubiquitinating activity-dependent and -independent manner, which is contrary to the current idea of ubiquitin-dependent sorting of proteins to exosomes. Copyright © American Society for Investigative Pathology.