The drug hydroxyurea (HU) is used for cancer therapy and treatment of sickle cell anemia. It inhibits cell cycle progression by blocking DNA synthesis and drives cells to undergo apoptosis or enter senescence. We demonstrate here that HU induces the expression of two AP-1 proteins, c-Jun and JunB, which exert antagonistic effects on the cell cycle. Moreover, the induction of c-Jun is observed following treatment with two other drugs that inhibit the cell cycle in S phase, aphidicolin and camptothecin. The induction of c-Jun, which promotes cell cycle progression, up-regulates expression of cyclinDafter exposure of cells to HU. Deficiency in c-jun prevents elevation of cyclin D expression and extends entrance into HU-induced senescence but also renders cells more resistant to HU-dependent apoptosis. The induction of c-Jun is independent of JNK activity, and additionally, of c-Jun autoregulatory activity but is inhibited upon inhibition of protein kinase C activity. Therefore, we suggest that c-Jun activity prevents drug-induced senescence. Conversely, the JunB target gene, tumor suppressor p16INK4a, a cyclin-dependent kinase inhibitor essential for the induction of drug-induced senescence, is also up-regulated by HU in a JunB-dependent manner. Constitutive expression of JunB up-regulates p16INK4a and increases the sensitivity of mouse fibroblasts to drug-induced-senescence. Thus, we suggest that in contrast to c-Jun, JunB drives cells to enter HU-dependent senescence. The effect ofHU treatment, which regulates the intricate web of AP-1 transcription, depends on the balance between c-Jun and JunB activities.