The genome of ovine adenovirus OAV287 has an arrangement which is unique among known adenoviruses. To facilitate further experimentation on the structure and function of this genome, plasmids containing a complete clone of the genome were constructed. The cloned viral genome was released from plasmids by restriction enzyme digestion as an intact linear molecule with authentic 5' termini. Transfection of the linear DNA into cells which supported replication produced infectious virus. Mutation of a unique SalI site at the right-hand end of the genome disrupted reading frames of unknown function without affecting virus rescue, identifying this region as nonessential for replication in vitro. A 20-bp oligonucleotide was also inserted into the short intergenic region between the pVIII and the fiber sequences, identifying a second site for gene insertion. These studies will facilitate the development of OAV as a gene transfer vector.