Neuropeptide Y (NPY) modulates cardiovascular, feeding and reproductive functions. Peripheral neurohumoral inputs from these systems are integrated and transformed into efferent signals in the brainstem. Detailed mapping of NPY-expressing cells in the brainstem has not been established in primates. In this report we utilized the in situ hybridization (ISH) method to identify brainstem areas that contain NPY mRNA in four ovariectomized rhesus macaques treated with estradiol-17beta. A 35S-labeled human NPY cRNA probe was used for ISH in paraformaldehyde-fixed brainstem blocks that were sectioned at 20 microm thickness. In the upper cervical spinal cord, NPY mRNA signals were confined to the substantia gelatinosa along the spinal tract of the trigeminal nerve. In the medulla, NPY images were found in the nucleus of solitary tract, dorsal motor nucleus of vagus nerve, nucleus of the spinal tract of trigeminal nerve, lateral reticular nucleus and the reticular formation. In the pons, NPY mRNA was confined to cells in the locus coeruleus and the nucleus of raphe. NPY signals were observed in the ventral portion of the periaqueductal grey, the dorsal nucleus of raphe and the reticular formation of mesencephalon in the midbrain. Whereas the brainstem distribution of NPY-containing cells in the rhesus macaque overlap those regions that are rich in catecholamines, NPY perikarya were also present in 'noncatecholaminergic' areas. For example, the reticular formation of both the medulla and mesencephalon abundantly expressed NPY mRNA. The functional significance of, and the effects of estrogen on, these patterns in NPY expression is unknown.