Human adenovirus proteinase (AVP) requires two cofactors for maximal activity, pVIc, a peptide derived from the C-terminus of adenovirus precursor protein pVI, and the viral DNA. Synchrotron protein footprinting was used to map the solvent accessible cofactor binding sites and identify conformational changes associated with the binding of cofactors to AVP. The binding of pVIc alone or pVIc and DNA together to AVP triggered significant conformational changes adjacent to the active site cleft sandwiched between the two AVP sub-domains. In addition, upon binding of DNA to AVP, specific residues on each of the two major sub-domains were observed to be significantly protected from hydroxyl radicals. Based on the locations of these protected side-chain residues and conserved aromatic and positively charged residues within AVP, a three-dimensional model of DNA binding was constructed. The model indicated that DNA binding can alter the relative orientation of the two AVP domains leading to the partial activation of AVP by DNA. In addition, both pVIc and DNA may independently alter the active site conformation as well as drive it cooperatively to fully activate AVP.