The striatum is a key structure of basal ganglia controlling extrapyramidal motor activity and processing addictive plasticity of abused substances. Glutamatergic transmission that is enriched in the striatum regulates a variety of striatal neuronal activities via selective activation of ionotropic and metabotropic glutamate receptors (mGluRs). In this study, the interaction between N-methyl-D-aspartate (NMDA) receptors and group I mGluRs (mGluR1 and mGluR5 subtypes) in activating a phosphorylation cascade to a transcription factor cAMP response element-binding protein (CREB) was investigated in primary cultures of E18 or postnatal day 1 striatal neurons. We found that activation of NMDA receptors with NMDA rapidly and concentration-dependently increased the number of neurons expressing phosphorylated CREB (pCREB) as revealed by immunocytochemistry. The increased pCREB expression by NMDA was sensitive to an NMDA antagonist MK801. Co-incubation of a subthreshold dose of a group I mGluR agonist 3,5-dihydroxyphenylglycine (DHPG) that itself did not alter basal pCREB expression augmented NMDA-induced CREB phosphorylation. The mGluR5 antagonist 2-methyl-6-(phenylethynyl)pyridine hydrochloride blocked the DHPG augmentation of NMDA-induced CREB phosphorylation, while the mGluR1 antagonist 7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester did not. Interestingly, the protein kinase C inhibitors chelerythrine and Gö6983 also prevented DHPG from enhancing CREB phosphorylation induced by NMDA. Whereas a low dose of the protein kinase C activator phorbol 12-myristate 13-acetate mimicked the DHPG potentiation. These results indicate a facilitatory regulation of an NMDA cascade to CREB phosphorylation by concurrent glutamatergic tone on mGluR5, which is probably processed via an intracellular signaling pathway involving protein kinase C.