The distribution of simian virus 40 large tumor antigen in subcellular fractions from simian virus 40-transformed hamster (H-50) and mouse (VLM) cells and from simian virus 40-infected monkey cells was determined. Solubilized [3S]methionineor 'Pi-labeled surface membrane and nuclear fractions were prepared, immunoprecipitated with hamster anti-T serum, and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Tumor antigen with an apparent molecular weight of -96,000 was detected in both subcellular fractions. Minor components of -68,000 and -56,000 with anti-T reactivity which labeled with [3S]methionine were also detected in both fractions from H-50 cells, as were components of -140,000 and -56,000 from VLM cells. The 56,000 component appeared to be greatly reduced in 32Pi-labeled surface membrane fractions. Normal cells or cells transformed with a heterologous agent, such as polyoma virus or a chemical carcinogen, lacked immunoprecipitable tumor antigen. Cell fractionation was monitored by [3H]thymidine labeling, NADH-diaphorase activity, and Na+-K+-dependent ATPase activity. These analyses revealed only trace contamination of surface membranes by nuclei, extremely low levels of nuclear rupture during homogenization, and an approximate 10-fold enrichment of surface membrane. Reconstruction experiments demonstrated that soluble tumor antigen failed to associate or copurify with surface membranes during fractionation procedures. These results indicate the presence of a protein in the plasma membrane of cells transformed or infected by simian virus 40 that is immunologically indistinguishable from nuclear tumor antigen. 523 on July 6, 2020 by guest http://jvi.asm.org/ Downloaded from 524 SOULE AND BUTEL Al was added to each antigen-antibody reaction. After incubation for 5 min at 0°C, the immune complexes were washed three times with BDM by centrifugation at 2,400 x g for 10 min. Antigen was eluted from the immune complex by resuspending the final bacterial pellet in 50 pl of electrophoresis sample buffer (0.0625 M Tris [pH 6.8], 2% SDS, 2% ,-mercaptoethanol, 5% glycerin, and 0.005% bromophenol blue) and boiling for 3 min. Bacteria were removed by centrifugation at 17,000 x g for 10 min at 4°C. The supernatant was carefully removed and subjected to SDS-gel electrophoresis on discontinuous 12.5% SDS-polyacrylamide slab gels (14 by 12 by 0.15 cm) as previously described (16,19,39,40), with acrylamide-methylene bisacryl-J. VIROL.on July 6, 2020 by guest