Mouse 3T3 cells contain low levels of a 54,000 molecular weight protein that is specifically immunoprecipitated by sera from animals bearing SV40-induced tumors. Following infection or transformation of 3T3 cells with SV40, the levels of the 54,000 MW protein increased 25- to 50-fold. In SV40-infected 3T3 cells the rate of synthesis of the viral 94,000 M WT antigen increased over the first 14 hr after infection and then declined. In contrast, the rate of synthesis and/or the stability of the 54,000 MW protein increased over a 22-hr period and then remained at maximal levels for an additional 30 hr. Temperature-sensitive A gene mutants of the SV40 large T antigen, at the nonpermissive temperature, failed to initiate or maintain the maximal increased levels of the 54,000 MW protein observed in virus-infected or -transformed cells. SV40 deletion mutants in the small t antigen unique region of the genome stimulated the production of wild-type virus levels of the 54,000 MW protein in virus-infected cells. The results of these experiments demonstrate that the SV40 A gene product is required to initiate and possibly maintain the high levels of the 54,000 MW protein found in virus-infected and -transformed cells. © 1979.