Cytoskeletal proteins are exploited by many viruses during infection. We report a novel finding that actin can act as a cofactor for the adenovirus proteinase (AVP) in the degradation of cytoskeletal proteins. Transfection studies in HeLa cells revealed AVP localized with cytokeratin 18, and this was followed by destruction of the cytokeratin network. For AVP to cleave cytokeratin 18, a cellular cofactor was shown to be required, consistent with AVP being synthesized as an inactive proteinase. Actin was considered a cellular cofactor for AVP, because the C terminus of actin is homologous to a viral cofactor for AVP. AVP was shown to bind to the C terminus of actin, and in doing so AVP exhibited full enzymatic activity. In vitro, actin was a cofactor in the cleavage of cytokeratin 18 by AVP. The proteinase alone could not cleave cytokeratin 18, but in the presence of actin, AVP cleaved cytokeratin 18. Indeed, actin itself was shown to be a cofactor and a substrate for its own destruction in that it was cleaved by AVP in vitro. Cleavage of cytoskeletal proteins weakens the structure of the cell, and therefore, actin as a cofactor may play a role in cell lysis and release of nascent virions.