We have analyzed the mechanisms used by adenovirus to gain entry into the host cell. Using both virus infection and the ability of adenovirus particles to enhance polylysine/DNA uptake as a measure of virus entry, we have demonstrated that the adenovirus-encoded 23K protease is required for two functions in the infection process. A proteolytic processing of the capsid is required to generate a virus capsid that can increase membrane interactions at pH 5. We have found that forms of adenovirus capsid that have not undergone the processing reactions (immature capsids) are deficient in their ability to disrupt membranes at pH 5 and are unable to enhance the entry of polylysine/DNA complexes. A second role of the protease was revealed by experiments using inhibitors of the protease. Mature virus capsids lose their ability to enhance gene delivery and become noninfectious after exposure to inhibitors of the protease (1 microM N-ethylmaleimide, 100-300 microM copper chloride, 1 microM MDL28170, or anti-protease antiserum), suggesting that the viral protease activity is required during the cellular entry process.