Immunohistochemical methods were used in the rat to plot the distribution of neuropeptide Y (NPY) immunoreactivity in the paraventricular (PVH) and supraoptic (SO) nuclei of the hypothalamus, and a combined retrograde transport-double immunohistochemical labeling technique was used to determine the extent to which NPY immunoreactivity is coexpressed in brainstem cell groups that stain with antisera to phenylethanolamine-N-methyltransferase (PNMT; a marker for adrenergic neurons) or dopamine-ß-hydroxylase (DBH; a marker for adrenergic and noradrenergic neurons) and also project to the PVH. The results confirm the existence of a major NPY-immunoreactive pathway that is in a position to influence each major class of output neurons in the PVH. Thus, most parts of the parvicellular division receive a dense input that is similar to, though somewhat more extensive than, the one stained by DBH antisera. However, in the magnocellular division catecholaminergic inputs are preferentially associated with vasopressinergic neurons, while NPY-stained fibers tend to be more evenly distributed in regions containing both oxytocinergic and vasopressingergic neurons, and their density appear to be lower than that of DBH-stained fibers. In the SO, only a moderate NPY-stained input was apparent, while, as described previously, DBH-immunoreactive fibers are rather dense and are preferentially distributed in vasopressinergic regions of the nucleus. The results of combined retrograde transport-double immunohistochemical labeling experiments may be summarized as follows: the vast majority of cells in the medulla that were retrogradely labeled after discrete implants of the fluorescent tracer true blue into the PVH, and were PNMT-immunoreactive, also stained for NPY. However, less extensive co-localization was detected in noradrenergic cell groups of the caudal medulla. About 60% of the retrogradely labeled-DBH positive cells in the Al cell group were also NPY-positive, while those in the caudal part of the nucleus of the solitary tract (the A2 cell group) usually failed to stain with anti-NPY. Similarly, in the locus coeruleus (the A6 cell group) where virtually all retrogradely labeled neurons were DBH-positive, only rarely were triply labeled cells detected. These results suggest that NPY immunoreactivity is extensively co-contained within adrenergic neurons of the C1, C2, and C3 groups that project to the PVH, while the correspondence in noradrenergic cell groups is less complete, and generally limited to a subset of neurons in the A1 cell group. Differences in the distribution of NPY- and DBH-stained inputs to the magnocellular division of the PVH and the SO suggest a complex arrangement of NPY-catecholamine colocalization in the A1 region. The staining patterns are consistent with the view that NPY is a neurotransmitter or neuromodulator of major importance in brainstem projections to the PVH and SO, and that by virtue of its distribution, NPY provides further differentiation of an elaborate system of ascending catecholaminergic inputs to the hypothalamus.