The hexon genes of human adenovirus (AV) types 3, 16 (both subgenus B), and 4 (subgenus E) were sequenced and the nucleotide and predicted amino acid sequences were compared. We observed a high degree of homology between the three serotypes and thus could confirm the close evolutionary relationship of subgenera B and E which was previously inferred after comparing other genome regions. Employing the three-dimensional model of the AV2 hexon, the structures of the three hexons were predicted. The major differences between the hexons of AV3/AV16 and AV3/AV4 were confined to the I1 and I2 surface loops. As expected from cross-reactions in neutralization, the hexons of AV16 and AV4 were not only very homologous in the conserved regions but also in the loops I1 and I2. This indicates that the type-specific antigenic determinant is located in these regions of the hexon. Closer examination of the loop I4 sequences, including also serotypes of subgenera A, C, and F, revealed that this region is subgenus-specific. The finding that AV4 differs from AV3 and AV16 in loop 1(4) supports the placement of AV4 in a separate subgenus.