Abstract: A ubiquitin-specific protease which cleaves ubiquitin from any protein or peptide to which ubiquitin is joined and the gene encoding the protease are disclosed. The protease specifically cleaves the peptide bond in a fusion of ubiquitin to a protein or peptide between the carboxyl-terminal amino acid residue of a ubiquitin moiety and the .alpha.-amino group of any non-ubiquitin protein or peptide to which ubiquitin is joined. Recombinant expression vectors containing a DNA sequence encoding the ubiquitin-specific protease can be used to transform cells for production of the protease or to provide the cell with the ability to proteolytically deubiquitinate, in vivo, ubiquitin fusions co-produced by the cell. The protease can also be isolated and used to deubiquitinate ubiquitin fusions in vitro.