Previously, we found that cytochrome oxidase-rich zones in the supragranular layers of the macaque striate cortex had more asymmetric, glutamate-immunoreactive synapses than the surrounding, cytochrome oxidase-poor regions. A major glutamate receptor family is N-methyl-D-aspartate, which is implicated in the stimulation of nitric oxide synthase and in the production of nitric oxide, a gaseous intra- and inter-cellular messenger. To determine if energy-generating and energy-utilizing enzymes bore any spatial relationship with neurochemicals associated with glutamatergic neurotransmission in the monkey visual cortex, serial cortical sections were processed histochemically for cytochrome oxidase and NADPH-diaphorase, and immunohistochemically for sodium/potassium-ATPase, nitric oxide synthase, and N-methyl-D-aspartate receptor subunit 1 protein, respectively. The general patterns were similar among the five neurochemicals, with layers 4C, 6 and supragranular puffs being labelled, although the intensity of labelling differed among them. Monocular impulse blockade with tetrodotoxin for two to four weeks induced a down-regulation of all five neurochemicals not only in deprived layer 4C ocular dominance columns, but also in deprived rows of puffs. Thus, the regulation of all five neurochemicals in the mature visual cortex is activity-dependent. Combined cytochrome oxidase histochemistry and nitric oxide synthase immunohistochemistry in the same sections revealed that double-labelled cells were primarily medium-sized non-pyramidals in various cortical layers. Likewise, those that were double-labelled by N-methyl-D-aspartate receptor subunit 1 immunohistochemistry and nitric oxide synthase immunogold silver staining in the same sections were of the medium-sized non-pyramidal neurons. At the ultrastructural level, combined cytochrome oxidase cytochemistry and postembedding immunogold labelling for nitric oxide synthase showed that immunogold particles for nitric oxide synthase were more heavily concentrated in cytochrome oxidase-rich type C cells. These medium-sized non-pyramidal cells were previously found to be gamma aminobutyric acid-immunoreactive and received both gamma aminobutyric acid- and glutamate-immunoreactive axosomatic synapses. Thus, our results are consistent with an enrichment of excitatory synaptic interactions in metabolically active regions of the primate visual cortex that involves glutamate-related neurochemicals, such as N-methyl-D-aspartate receptors and nitric oxide synthase. These interactions impose a higher energy demand under normal conditions and are down-regulated by retinal impulse blockade.