Protein kinases are thought to play a key role in signal transduction and oncogenesis, but little is known about the intranuclear phosphorylation events associated with transformation. Here we report on cell cycle-dependent phosphorylation of cytoskeleton-associated 350 kD protein and the regular interchange in its location between the nucleus and cytoplasm of normal cells. Persistent intranuclear location of the phosphorylated 350 kD protein was also found throughout the cell cycle in transformed cells, as detected by immunoprecipitation of 32P-phosphorylated 350 kD protein from isolated nuclei and immunofluorescent staining with a monoclonal antibody that recognized phosphorylated site of 350 kD protein. A conditional transformed phenotype induced by a temperature-sensitive (ts) viral oncogene or a transforming growth factor was also associated with the intranuclear presence of the phosphorylated 350 kD protein. Thus the 350 kD protein seems to be a target molecule of protein kinases that are stimulated directly or indirectly by growth factors or by oncogene products in the nucleus, and appears to be a new transformation-related nuclear antigen.