Ovine adenovirus OAV287 (OAV) is phylogenetically and serotypically distinct from human Ad5. OAV grows productively in CSL503 foetal ovine lung cells and abortively infects several human cell lines. OAV has a unique fiber and a penton protein that lacks a recognisable integrin-binding motif. It is not known whether a secondary receptor is required for infection. A hybrid virus was constructed in which the cell binding domain on the OAV fiber protein was exchanged for the equivalent region from human adenovirus type 5. The hybrid OAV grew to titres that were 1 to 2 log10 lower than wild-type OAV in permissive ovine cells. Human Ad5 also infected CSL503 cells but failed to compete with OAV for receptor binding sites on those cells. However, the hybrid virus did compete with Ad5, consistent with its use of the Ad primary receptor. The hybrid virus was also neutralised by Ad5 antiserum whereas OAV was not. Human 293 kidney and LNCaP prostate cell lines that were not detectably infected by OAV were infected by the hybrid virus and other human prostate and breast cancer cell lines showed greatly enhanced infectivity. Thus, modification of the fiber cell binding domain was sufficient to profoundly alter the tropism of OAV, suggesting that the interaction between the primary receptor and the virus particle is the major factor controlling virus entry during infection.