We introduced into tk- human 143 cells adenovirus type 2 (Ad2) genes by transformation with a plasmid (p711) containing both Ad2 sequences and the herpes simplex virus type 1 (HSV-1) tk gene. p711 contained approximately the left 8% of the Ad2 genome inserted in the HindIII site of pBR322, whereas the fragment of HSV-1 containing the tk gene was inserted in the BamHI site. Three tk+ cell lines were isolated after selection in HAT medium. The arrangement of viral sequences in the three transformants was analyzed by restriction endonuclease digestion and filter hybridization. All three lines contained a single insertion of Ad2 DNA which was present at approximately one copy per cell. The arrangement of Ad2 sequences in these lines was identical to that found in the linear p711 DNA used in the transformation. S1 analysis of the Ad2-specified RNA from two of these lines indicated that the early region 1a mRNA's were synthesized, though in lower amounts than found in lytic infections. These cell lines contained only the left half of early region 1b (4.6 to 11.2), which encoded the 5' portion of the 1b mRNA's. A complex pattern of 1b RNAs was made in these cell lines. Transcription of most of these RNAs began at or near the 1b promoter and proceeded through the 1b sequences into the flanking pBR322, HSV-1, or host sequences. Since many of the RNAs were terminated or spliced in the HSV-1 (anti-sense strand) or pBR322 sequences, new RNA processing sites must be used in the formation of these mRNA's. All three lines fully complemented the 1a deletion mutant Ad5 dl312. Surprisingly, these lines also permitted the growth of 1b deletion mutants (Ad5 dl313 and Ad5 dl434), although the complementation was not always complete. Presumably the new gene product(s) which contained only part of the 1b gene provided most of the essential function(s) required for viral multiplication. Alternatively, the 1b 19-kilodalton protein which was entirely encoded by the adenovirus sequences present in these cell lines was sufficient for viral growth even in the absence of the 1b 55-kilodalton protein.