Approximately 2 kilobases (kb) of additional DNA can be packaged into wild-type virions of human adenovirus type 5 (Ad5). To extend this limit, a helper independent Ad5 cloning vector was constructed by deleting most of early region 3 (E3) from map coordinates 78.5 to 84.7 and essentially all of early region 1 (E1) from coordinates 1.0 to 10.6. E3 is nonessential for adenovirus replication in cultured cells, and E1 is nonessential when the virus is propagated in 293 cells which constitutively express the E1 gene products. The resulting new virus, dlE1,3 is about 5.5 kb shorter than wild-type Ad5 and therefore should be able to accept up to 7.5 kb in foreign DNA. To test the usefulness of this vector, the herpes simplex virus type 1 (HSV-1) thymidine kinase gene (tk) along with its regulatory sequences was inserted into the unique XbaI site of dlE1,3 (at map position 78.5/84.7). The resulting recombinant virus, Adtk, expressed the HSV tk at a low level (as compared with HSV-1) in infected cells; however, tk expression was markedly enhanced when Adtk-infected cells were superinfected with a tk- mutant of HSV. Furthermore, the Adtk virus efficiently transformed tk- mouse cells (line LTA) to the tk+ phenotype. At a low efficiency, it was also possible to transform tk- human cells (line 143), and tk+ transformants of both mouse and human origin have been established as permanent lines.