An bovine adenovirus, which is phylogenetically distinct from the Mastadeno- and Aviadenoviruses, was used to construct recombinants in which reporter genes were expressed from the OAV major late, or human cytomegalovirus promoters. It was demonstrated by transgene expression that OAV could infect bovine nasal turbinate and rabbit kidney cells as well as a range of human cell types, including lung and foreskin fibroblasts as well as liver, prostate, breast, colon, and retinal lines. Some human lines, e.g., 293 and LNCaP were not detectably infected. Infection occurred even though OAV has a fiber protein with a unique cell binding domain and a penton protein that lacks the integrin-binding Arg-Gly-Asp motif which facilitates entry by human adenoviruses. Most cell lines showed little or no ill effect for several days after infection but a prominent cytopathic effect appeared in fibroblasts after 3-4 days. However, no viral DNA synthesis was detected and replication was abortive. Viral promoter activity during infection of nonpermissive cell types was assayed by RT-PCR. Early promoter activity was detectable in some, but not all cell types. In a liver and a colon carcinoma cell line, none of the promoters examined was significantly active, even when a higher multiplicity of infection was used. Major late promoter activity was not detectable in any cell type. The lack of DNA replication and MLP function suggests that a critical transition from early to late gene expression does not occur during abortive infection by OAV.