Cell cycle-dependent phosphorylation and nuclear import of the tumorigenic transcription factor viral Jun (v-Jun) were investigated in chicken embryo fibroblasts. Nuclear accumulation of v-Jun but not of cellular Jun (c-Jun) is cell cycle dependent, decreasing in G1 and increasing in G2. The cell cycle-dependent regulation of v-Jun was mapped to a single serine residue at position 248 (Ser248), adjacent to the nuclear localization signal (NLS). Ser248 of v-Jun represents an amino acid substitution, replacing cysteine of c-Jun. It was shown by peptidase digestion and immunoprecipitation with antibody to the NLS that v-Jun is phosphorylated at Ser248 in the cytoplasm but not in the nucleus. This phosphorylation is high in G1 and low in G2. Nuclear accumulation of v-Jun is correlated with underphosphorylation at Ser248. The regulation of nuclear import by phosphorylation was also examined using NLS peptides with Ser248 of v-Jun. Phosphorylation of the serine inhibited nuclear import mediated by the NLS peptide in vivo and in vitro. The protein kinase inhibitors staurosporine and H7 stimulated but the phosphatase inhibitor okadaic acid inhibited nuclear import mediated by the NLS peptide. The cytosolic activity of protein kinases phosphorylating Ser248 increased in G0 and decreased during cell cycle progression, reaching a minimum in G2, whereas phosphatase activity dephosphorylating Ser248 was not changed. These results show that nuclear import of v-Jun is negatively regulated by phosphorylation at Ser248 in the cytoplasm in a cell cycle-dependent manner.