Microtubule-associated protein MAP-2 is a neuronal phosphoprotein which modulates microtubule stability and spatial organization of signal transduction pathways. The functions of MAP-2 are modulated by phosphorylation. We studied the modulation of MAP-2 phosphorylation using the N-methyl-d-aspartate (NMDA) type of glutamate receptors and the signal transduction pathways mediating this modulation in primary cultures of rat cerebellar neurons. NMDA induced a rapid increase (330% of basal at 5 min) in MAP-2 phosphorylation which was not prevented by KN-62, indicating that it is not mediated by activation of Ca-calmodulin-dependent protein kinase. NMDA-induced phosphorylation of MAP-2 was inhibited by the nitric oxide synthase inhibitors nitroarginine and 7-nitroindazole and by PD098059 (an inhibitor of MAP kinase kinase), but was only slightly reduced by calphostin C or U-73122, inhibitors of protein kinase C and of phospholipase C, respectively. This indicates that the main pathway mediating NMDA-induced phosphorylation of MAP-2 is activation of nitric oxide synthase and subsequent activation of MAP kinase. We show that activation of NMDA receptors induces an activation of MAP kinase which is prevented by nitroarginine. The nitric oxide-generating agent (±)-S-nitroso-N-acetylpenicillamine (SNAP) also induced activation of MAP kinase and increased phosphorylation of MAP-2. Other nitric oxide-generating agents (NOC-18 and NOR-3) also increased MAP-2 phosphorylation. The interplay between NMDA receptors-associated signal transduction pathways and MAP-2 may be involved in the modulation of neuronal responses to extracellular signals and in the regulation of neuronal function.