Simple methods are described for expressing the endoproteinase polypeptides of adenovirus serotypes 2 and 12 in Escherichia coli and for purifying these products from crude bacterial extracts using immobilized metal affinity chromatography. A plasmid for expressing an artificial adenovirus substrate that can be purified by the same method also is described. Cleavage of this substrate by the Ad2 virion proteinase confirmed that the cleavage specificity of the adenovirus proteinase is determined by the four amino acids immediately before the cleavage site. The purified recombinant Ad2 endoproteinase alone was incapable of cleaving the artificial substrate, but cleavage occurred if the artificial substrate was incubated with both recombinant endoproteinase and H2ts1 virions or heat-inactivated wild-type Ad2 virions. These results indicate that, in addition to the 23-kDa proteinase polypeptide, cofactors present in Ad2 virions are required to produce active adenovirus proteinase.