Anatomical and pharmacological evidence suggests a role for substance P (SP) in the control of vasopressin secretion, but the origins of SP-immunoreactive (IR) projections to the paraventricular (PVH) and supraoptic (SO) nuclei of the hypothalamus have not yet been identified. Combined axonal transport, immunohistochemical, and ablation approaches were used to characterize the organization of SP-IR projections to the PVH. The results may be summarized as follows: (1) SP-IR projections are broadly and prominently distributed throughout the SO and both the magnocellular and parvicellular divisions of the PVH. The distribution within the PVH is quite uniform. (2) Combined retrograde transport-immunohistochemical analyses identified multiple potential sources of SP-IR inputs to the PVH. These included a number of hypothalamic cell groups, the laterodorsal and peduculopontine tegmental nuclei, and the rostral and caudal aspects of the ventrolateral medulla. Portions of the tegmental and medullary SP-IR neurons that were retrogradely labelled following tracer deposits in the PVH also stained positively for choline acetyltransferase or tyrosine hydroxylase, respectively. (3) To evaluate the distribution and prominence of medullary SP-IR projections to the PVH and SO, staining for SP and catecholamine-synthesizing enzymes was carried out in animals that had previously received knife cuts at the level of the pontomedullary border. Pronounced, and roughly parallel decrements in staining for peptide and amines were seen in the magnocellular division of the PVH and in the SO; less marked reductions in SP-IR varicosities are in a position to influence multiple visceral regulatory cell types in the PVH and SO. Inputs to the magnocellular neurosecretory system arise in large measure from medullary neurons in which SP coexists with catecholamines. SP-IR projections to the parvicellular division of the PVH appear to originate from a number of sources.