The nature of the infection of mouse B3T3 cells by adenovirus type 2 (Ad2) has been studied in vitro. Following infection with an adsorbed MOI of 225, more than 90 percent of the cells synthesized both early and late virus-specific antigens. In contrast, the yield of progeny virus varied from only 2 X 10(4) to 2 X 10(6) FFU/2 X 10(5) cells. The range in yields was related, in part, to the number of cell generations from the time of the initial subcloning, the yield increasing with passage level. Infectious center analysis suggested that fewer than 0.5 percent of infected cells synthesized progeny virus. Analysis of DNA synthesis in infected multiplying B3T3 cells demonstrated that cellular DNA synthesis began to be shut off at 12 hours p.i., a time when viral DNA synthesis was beginning. The maximum rate of viral DNA synthesis was approximately 12 percent of that in infected human cells. In contrast to infected multiplying cells, infection of quiescent B3T3 cell cultures resulted in the induction of cellular, along with viral, DNA synthesis. Analysis of late gene expression detected synthesis of most viral polypeptides, but revealed greater than 90 percent reductions in the rate of synthesis of polypeptides II, III, IV, and IX, as compared with infected human cells.