Monospecific antibodies to glutamate were used to characterize the organization of excitatory neurons and the plasticity of glutamate expression in the macaque striate cortex. Somata and processes immunoreactive for glutamate were densely and unevenly distributed in layers II-III, IVA, IVC. In tangential sections through layers II and III, patches of intense glutamate immunostaining were observed and were found to coincide with regions of the cytochrome oxidase (CO)-rich puffs. By contrast, clusters of intense immunostaining were surrounded by the lightly immunostained but intensely CO-stained lattice in layer IVA. Similarly, in layer IVC, focal aggregates of intense glutamate immunoreactivity were interspersed among regions of light immunostaining but intense CO staining. Glutamate immunoreactivity was also intense in layer VI but was much lighter in layers I, IVB, and V. Throughout the striate cortex, neurons resembling pyramidal cells and spiny stellate cells and processes that included dendrites and axons were immunostained. None of the glutamate-positive neurons was GABA immunoreactive. Following monocular deprivation of adult monkeys by intravitreal injections of TTX into one eye, glutamate immunoreactivity in layers IVC was distributed in alternating intensely and lightly stained stripes. The stripes of reduced immunostaining, which contained an abnormally low concentration of glutamate neurons and pale neuropil, corresponded to columns dominated by the TTX-injected eye. Similar stripes of alternating intense and light immunoreactivity were seen in layers II-III, where they corresponded to rows of puffs at the centers of intact-eye and deprived-eye columns, respectively. These findings demonstrate that glutamate-immunoreactive neurons and terminals in monkey striate cortex are densely concentrated in layers receiving direct geniculocortical innervation. In addition, the glutamate neurons and terminals form discrete units, which in layers II and III coincide precisely with regions receiving geniculocortical terminations but in layers IVA are segregated from these terminations. The findings also indicate that glutamate immunoreactivity is regulated by visually driven activity, and suggest that monocular deprivation in adulthood leads to a reduction in the major excitatory neurotransmitter in visual cortex as well as previously indicated reductions in GABA, the major inhibitory neurotransmitter.