All known human adenoviruses are classified as mastadenoviruses, while the ovine adenovirus (OAdV) serotype 7 is the prototype of the atadenoviruses, a proposed new genus. OAdV replicates abortively in human cell types and has potential as a gene transfer vector. However, the function of OAdV nonstructural genes is poorly understood and it is unclear whether OAdV replication might be complemented by a replicating human AdV in coinfected cells. To investigate possible interactions three human cell lines were singly infected with OAdV or human AdV5 or doubly infected. The development of a cytopathic effect and genome replication was monitored over three passages in each cell type. No significant OAdV replication occurred in any of the cell types examined either in the presence or in the absence of replicating AdV5. No aberrant AdV5 genome products were detected in coinfected cells. In contrast, in coinfected cells an OAdV recombinant that expressed the AdV5 E1A gene was able to promote the replication of an AdV5 E1A-deficient mutant, demonstrating trans-complementation between appropriate viruses. These findings have implications for the biosafety of OAdV vectors and their possible utility for enhancing gene delivery.