SV40 transformed mouse cells (mKSA) were labeled in parallel with either [35S]methionine or [3H]palmitate and subfractionated. Nuclear extracts and solubilized plasma membranes were analyzed for the presence of either 35S- or 3H-labeled SV40 large tumor antigen by immunoprecipitation and SDS polyacrylamide gel electrophoresis. The majority of the [35S]methionine labeled large T was recovered from the nuclear fraction, only minor amounts were detected in plasma membranes. In contrast, large T labeled specifically with [3H]palmitate was found only in the plasma membrane fraction. Our results demonstrate a specific acylation of large T associated with plasma membranes, suggesting that the membrane location of this predominantly nuclear protein is specific.