Full-length Ad2 penton base (PbFL571) and amino-terminal (PbAT69) and carboxy-terminal deletion mutants (PbCT550 and PbCT531) were expressed at high levels in recombinant baculovirus-infected insect cells. PbFL571, and with a lesser efficiency PbAT69, assembled penton base capsomers (9S pentamers), whereas pentamer assembly was abolished with the deletion of the 21 C-terminal amino acids (as in PbCT550). A discrete population of penton base capsomers in PbFL571 and PbAT69 was found to occur as 12S decamers. PbFL571 and PbAT69 penton base were able to bind to full-length (but not to N-terminal deletion mutants of) fiber trimer to form authentic penton capsomer when coexpressed in trans in double-infected cells. Penton base monomers accumulated by PbCT550 and PbCT531 were capable of interacting with both fiber trimers and monomers. This suggested that mutual binding sites exist in the fiber and penton base subunits, and that the fiber-binding domain is located between residues 70 and 531 in the penton base, with its complementary domain situated at the N-terminus of the fiber. Intranuclear inclusions of recombinant protein were observed with the three deletion mutants PbAT69, PbCT550, and PbCT531, implying the existence of karyophilic signal(s) in the central domain of penton base.