Dopamine- and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32) plays a central role in medium spiny neurons in the neostriatum in the integration of various neurotransmitter signaling pathways. In its Thr-34-phosphorylated form, it acts as a potent protein phosphatase-1 inhibitor, and, in its Thr-75-phosphorylated form, it acts as a cAMP-dependent kinase inhibitor. Here, we investigated glutamate-dependent signaling cascades in mouse neostriatal slices by analyzing the phosphorylation of DARPP-32 at Thr-34 and Thr-75. Treatment with glutamate (5 mM) caused a complex change in DARPP-32 Thr-34 phosphorylation. An initial rapid increase in Thr-34 phosphorylation was NMDA/alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/metabotropic glutamate-5 receptor-dependent and was mediated through activation of a neuronal nitric oxide synthase/nitric oxide/cGMP/cGMP-dependent kinase signaling cascade. A subsequent decrease in phosphorylation was attributable to activation of an NMDA/AMPA receptor/Ca2+/protein phosphatase-2B signaling cascade. This decrease was followed by rephosphorylation via a pathway involving metabotropic glutamate-5 receptor/phospholipase C and extracellular receptor kinase signaling cascade. Treatment with glutamate initially decreased Thr-75 phosphorylation through activation of NMDA/AMPA receptor/Ca2+/protein phosphatase-2A signaling. Thereafter, glutamate slowly increased Thr-75 phosphorylation through activation of metabotropic glutamate-1 receptor/phospholipase C signaling. Our analysis of DARPP-32 phosphorylation in the neostriatum revealed that glutamate activates at least five different signaling cascades with different time dependencies, resulting in complex regulation of protein kinase and protein phosphatase activities.