A variety of recombinant proteins derived from protein pVI of human adenovirus type 2 (Ad2) were analysed for their ability to bind Ad2 hexon in vitro. As pVI is also required for activation of the adenovirus-coded protease, the same pVI derivatives were assessed for their ability to activate recombinant adenovirus-coded 23K protease. Two regions, between amino acid residues 48-74 and 233-239 of pVI, were required for the interaction with hexon. These regions are highly conserved amongst mastadenovirus pVI proteins. Both these regions are capable on their own of binding hexon weakly but must be provided in cis for strong hexon binding. In addition, we found evidence to indicate than conformation as well as sequence was important for good hexon binding in our assays. Authentic processing of the appropriate recombinant pVI derivatives, by the recombinant protease, was obtained without the addition of other cofactors. These findings are discussed in relation to the role of pVI in triggering the adenovirus maturation pathway.