All herpesviruses contain a ubiquitin (Ub)-specific cysteine protease domain embedded within their large tegument protein, based on homology with the corresponding sequences of UL36 from herpes simplex virus type 1 and M48 from murine cytomegalovirus. This type of activity has yet to be demonstrated for cells infected with a gammaherpesvirus. By activity-based profiling, we show that the large tegument protein of murine gammaherpesvirus (MHV-68) ORF64 (273 kDa) is a functional deubiquitinating protease, as assessed by tandem mass spectrometry of adducts in extracts from MHV-68-infected cells that had been labeled with ubiquitin vinylmethylester, a ubiquitin-based active site-directed probe. The recombinantly expressed amino-terminal segment of ORF64 displays deubiquitinating activity toward Ub C-terminal 7-amido-4-methylcoumarin in vitro. The findings reported here for MHV-68 ORF64 extend those made for the alpha- and betaherpesvirus families and are consistent with an important, conserved enzymatic function of the tegument protein.