The linkage of the N-methyl-D-aspartate (NMDA) subtype of L-glutamate receptor to the nitric oxide (NO)/3, 5'-cyclic guanosine monophosphate (cGMP) intracellular signalling system was investigated in murine neocortical cultures by examining the effects of NMDA antagonists, NO synthase inhibitors, and drugs targeting second messenger systems on NMDA-stimulated synthesis of cGMP. NMDA-stimulated synthesis of cGMP was time- and concentration-dependent, and inhibited by competitive (LY 274614, 100 mu M) and non-competitive NMDA antagonists (MK-801 30 mu M, 7-chlorokynurenate 100 mu M, and ifenprodil 100 mu M). NO synthase inhibitors (NG-nitro-L-arginine, KN-62, diphenyleneiodonium) and LY 83583, an inhibitor of guanylate cyclase, all inhibited NMDA-stimulated cGMP synthesis in a concentration-dependent manner, demonstrating its dependence on the two enzymes. Phorbol 12-myristyl 13-acetate (0.1 mu M), arachidonic acid (1 mu M), and thapsigargin (10 mu M) produced approximately 50% inhibition of NMDA-induced cGMP synthesis. These observations demonstrate that all domains of the NMDA receptor-complex and of NO synthase are active in neocortical neuronal cultures, and that the essential NO/cGMP signalling system has complex interactions with other second messengers.