L-Glutamate and norepinephrine are examples of a major excitatory neurotransmitter and a neuromodulator in the cerebral cortex, respectively. Little is known of how chemical signaling between the anatomically distinct chemical pathways occurs. Specific activation of the N-methyl-D-aspartate (NMDA) class of glutamate receptor in synaptosomal preparations from guinea pig cerebral cortex caused release of both of these chemicals, and this release was blocked by agents that inhibit nitric oxide (NO) production or remove NO from the extracellular space. Furthermore, neurotransmitter release correlated with cortical NO production after NMDA receptor stimulation. These results suggest that NO production and its extracellular movement may be links in the pathway from NMDA receptor activation to changes in chemical signaling in surrounding synaptic terminals in the cerebral cortex.