The distribution of glutamate and neuronal nitric oxide synthase in the rat nucleus tractus solitarii was investigated by double fluorescent immunohistochemistry combined with confocal laser scanning microscopy. Cells and fibers that exhibited neuronal nitric oxide synthase immunoreactivity alone, glutamate immunoreactivity alone or both immunolabels were present in all subnuclei of the nucleus tractus solitarii, but staining intensities differed between the subnuclei. The percentages of double-labeled glutamate-immunoreactive cells also differed between the subnuclei. The central subnucleus contained the highest percentage of double-labeled glutamate-immunoreactive cells and the medial subnucleus contained the lowest. The percentages of double-labeled neuronal nitric oxide synthase-immunoreactive neurons likewise differed between the subnuclei. The central subnucleus contained the highest percentage of double-labeled neuronal nitric oxide synthase-immunoreactive neurons and the commissural subnucleus contained the lowest. Because of our interest in cardiovascular regulation, the anatomical relationship between glutamate-immunoreactive and neuronal nitric oxide synthase-immunoreactive fibers in the dorsolateral and commissural subnuclei was further examined at higher magnification. Close appositions were observed between neuronal nitric oxide synthase-immunoreactive and glutamate-immunoreactive fibers, between double-labeled and glutamate-immunoreactive fibers, and between neuronal nitric oxide synthase-immunoreactive and double-labeled fibers. We recognized that a single visual perspective might cause labeled fibers that pass in close proximity to appear to make contact. Therefore, we constructed three-dimensional images from serial optical sections obtained from the dorsolateral and commissural subnuclei by means of a confocal scanning microscope. Rotation of the three-dimensional images caused some fibers that had seemed to be in close apposition to other structures to separate from those structures. In contrast, some glutamate-immunoreactive and some neuronal nitric oxide synthase-immunoreactive fibers remained in close apposition regardless of the angle at which they were viewed. This study supports there being an anatomical link between glutamatergic and nitroxidergic systems in the nucleus tractus solitarii. Recognized physiological interactions between the two systems could occur through such a link.