Human adenovirus type 5 and temperature-sensitive mutants ts36, ts37, and ts125 induced cellular DNA synthesis in quiescent rodent cells at both permissive and nonpermissive temperatures. Cellular DNA synthesis induced by adenovirus type 5 or by serum required protein synthesis for both initiation and continuation, whereas viral DNA synthesis was not dependent upon continued protein synthesis once it was initiated. Both cellular and viral DNA replication was induced in adenovirus type 5-infected cells in the presence of dibutyryl cyclic AMP at concentrations which inhibited induction by serum which suggested that some of the controls of DNA synthesis in serum-treated and virus-infected cells are different. After adenovirus infection of quiescent cells, there was a decrease in the number of cells with G1 DNA content and an increase in cells with G2 diploid and greater DNA contents. Thus, adenovirus type 5 induces a complete round of cellular DNA replication, but in some cells, it induces a second round without completion of a normal mitosis. These results suggest that adenovirus type 5 is able to alter cell growth cycle controls in a way which may be related to its ability to transform cells.