JunD has been implicated as a negative regulator of cell proliferation. If and how JunD is regulated by growth factors however has not been well investigated. We now report that in quiescent murine 3T3T cells, JunD is present in a hypophosphorylated form, but that when quiescent cells are stimulated to proliferate with serum, JunD undergoes a transient increase in its phosphorylation that occurs within 10 min and persists for up to 4 h. The increase in JunD phosphorylation correlates with the induction of cell proliferation since only those growth factors that promote cell proliferation can induce JunD phosphorylation. Treatment of quiescent 3T3T cells with serum also induces significant decreases in JunD/AP-1 DNA binding activity within 2 h and in JunD expression after 8-48h. However, both hypophosphorylated and hyperphosphorylated forms of JunD can bind AP-1 DNA. These results suggest that serum growth factors can modulate JunD characteristics in a variety of apparently independent ways to overcome its negative regulatory effect in controlling cell proliferation. These include induction of a transient increase in JunD phosphorylation, repression of JunD AP-1 DNA binding activity and downregulation of JunD expression.