Western blot analysis with monoclonal antibodies, produced in response to immunization with gradient-purified adenovirus 41 (Ad41) virions, identified two epitopes of interest on protein VI of enteric adenoviruses. One epitope is unique to subgenus F adenoviruses (Ad40 and Ad41); the other epitope is common to subgenus A (Ad12, 18 and 31) and subgenus F(Ad40, 41) adenoviruses but is not shared by representative serotypes of subgenera B (Ad3 and 7), C(Ad1, 2 and 5), D(Ad8) or E (Ad4). Alignment of the deduced amino acid sequence of the genes encoding the protein VI precursor (pre-VI) of Ad40 and Ad41 (subgenus F), Ad12 and Ad31 (subgenus A), Ad2 and Ad5 (subgenus C) shows that the N-terminal one-third and C-terminal 23 amino acids of pre-VI are highly conserved. Within the central domain, pre-VI of subgenus F serotypes is more closely related to that of subgenus A serotypes than to pre-VI of the non-enteric subgenus C adenoviruses (Ad2 and Ad5). By expressing random oligonucleotide fragments of the Ad41 protein VI gene as part of a T7 gene 10 fusion protein, the two epitopes of interest were mapped to within the same 14 amino acid region in the central domain of protein VI. Given the association of subgenera A and F adenoviruses with paediatric gastroenteritis, the epitope shared by these serotypes may be functionally significant with respect to gut tropism. In addition, this epitope is potentially valuable as a target for the detection of enteric adenoviruses in clinical specimens.