The penton base gene from adenovirus type 12 (Ad12) was sequenced and encodes a 497-residue polypeptide, 74 residues shorter than the penton base from Ad2. The Ad2 and Ad12 proteins are highly conserved at the amino- and carboxy-terminal ends but diverge radically in the central region, where 63 residues are missing from the Ad12 sequence. Conserved within this variable region is the sequence Arg-Gly-Asp (RGD), which, in the Ad2 penton base, binds to integrins in the target cell membrane, enhancing the rate or the efficiency of infection. The Ad12 penton base was expressed in Escherichia coli, and the purified refolded protein assembled in vitro with Ad2 fibers. In contrast to the Ad2 penton base, the Ad12 protein failed to cause the rounding of adherent cells or to promote attachment of HeLa S3 suspension cells; however, A549 cells did attach to surfaces coated with either protein and pretreatment of the cells with an integrin alpha v beta 5 monoclonal antibody reduced attachment to background levels. Treatment of HeLa and A549 cells with integrin alpha v beta 3 or alpha v beta 5 monoclonal antibodies or with an RGD-containing fragment of the Ad2 penton base protein inhibited infection by Ad12 but had no effect on and in some cases enhanced infection by Ad2. Purified Ad2 fiber protein reduced the binding of radiolabeled Ad2 and Ad12 virions to HeLa and A549 cells nearly to background levels, but the concentrations of fiber that strongly inhibited infection by Ad2 only weakly inhibited Ad12 infection. These data suggest that alpha v-containing integrins alone may be sufficient to support infection by Ad12 and that this pathway is not efficiently used by Ad2.