Adenovirus-polyomavirus recombinant viruses were constructed in vitro by inserting a hybrid transcription unit composed of the adenovirus type 2 major late promoter and the early coding region of polyomavirus into the adenovirus type 5 vector Ad5 delta E1/dl309. The vector lacks the E1a and E1b transcription units and contains a unique restriction endonuclease cleavage site in their place. The polyomavirus genomic insert contained a small deletion which precluded the synthesis of functional small and middle T antigen but allowed for the synthesis of large T antigen. One recombinant virus, Ad5PyR39, which contained the hybrid transcription unit in the opposite transcriptional orientation from the overall direction of late-gene transcription, was studied in detail. Ad5PyR39 replicated efficiently without a helper virus in human 293 cells and expressed hybrid mRNAs of the expected size and composition that were translated to yield large T antigen. The large T antigen synthesized in 293 cells was the same size as that produced in mouse 3T6 cells lytically infected with polyomavirus, and this protein bound efficiently and specifically to the large-T-antigen-binding sites in polyomavirus DNA. Moreover, the large T antigen encoded by the recombinant virus proved capable of catalyzing the replication in mouse 3T6 cells of a plasmid containing the polyomavirus origin for DNA replication. Comparison of the amount of large T antigen produced in 3T6 cells infected with polyomavirus with that in 293 cells infected with Ad5PyR39, under optimal conditions for each system, revealed at least a fivefold greater yield of the protein on a per cell basis in the latter system compared with the former. Ad5PyR39 should prove to be useful to isolate large quantities of functional polyomavirus large T antigen for structural and biochemical studies.