Stable rat pituitary tumor cell lines expressing two isoforms of the dopamine D2 receptor, D2L (long) and D2S (short) (the GH3D2L and GH3D2S cell lines, respectively), were established, and the signaling pathway underlying the anti-proliferative and cell death effects of dopaminergic agonists was examined in these cells. After either dopamine or quinpirole treatment, the cell viability decreased significantly only in GH3D2L cells and GH3D2S cells, but not in GH3 cells where D2 receptors are absent. Treatment with haloperidol, a specific D2 receptor antagonist, rescued the dopamine-mediated decreased cell viability in both the GH3D2L and GH3D2S cells. Treatment of these cells with dopamine decreased the DNA synthesis rate, as demonstrated by the incorporation of 5-bromo-2'-deoxyuridine (BrdU). Dopamine-induced cell death was observed in the GH3D2L and GH3D2S cells, and was accompanied by DNA laddering and caspase-3 activation, which were blunted by haloperidol, indicating that dopamine-induced cell death in these cells is mediated by the dopamine D2 receptors. D2 receptor-mediated cell death in these cells correlated with the sustained and enhanced activation of p38 mitogen-activated protein kinase (MAPK) and the extracellular-signal regulated kinase (ERK)1/2 pathways. Treatment with SB203580, which is a specific p38 MAPK inhibitor and PD98059, which is an inhibitor of MEK1/ERK signaling, selectively abrogates dopamine-induced cell death. It was further shown that p38 MAPK and ERK activation was inhibited by the antioxidant, N-acetylcysteine (NAC), and that a treatment with haloperidol completely blocked the p38 and ERK activation induced by dopamine. These results suggest that dopamine induces an anti-proliferative effect and cell death via the dopamine D2 receptors, by means of the p38 MAPK and ERK pathways involving oxidative stress, in the pituitary tumor cells.