The hypothalamus provides a major projection to the spinal cord that innervates primarily lamina I of the dorsal horn and the sympathetic and parasympathetic preganglionic cell columns. We have examined the chemical organization of the neurons that contribute to this pathway by using combined retrograde transport of fluorescent dyes and immunohistochemistry for 15 different putative neurotransmitters or their synthetic enzymes. Our results demonstrate that 5 cytoarchitectonically distinct cell groups in the hypothalamus contribute to the spinal projection and that each has its own predominant chemical types. In the paraventricular nucleus, substantial numbers of hypothalamo-spinal neurons stain with antisera against arginine vasopressin (25-35%), oxytocin (20-25%), and met-enkephalin (10%). About 25% of the neurons with spinal projections in the retrochiasmatic area stain with an antiserum against alpha-melanocyte-stimulating hormone. Nearly 100% of the hypothalamo-spinal neurons in the tuberal lateral hypothalamic area stain with this same antiserum, but these cells do not stain for other proopiomelanocortin-derived peptides, and so probably contain a cross-reacting peptide. This population must be distinguished from an adjacent cell group, in the perifornical region, where many spinal projection neurons stain with antisera against dynorphin (25%) or atrial natriuretic peptide (20%). Finally, in the dorsal hypothalamic area as many as 55-75% of the neurons with spinal projections are dopaminergic, on the basis of their staining with an antiserum against tyrosine hydroxylase. These 5 neurochemically distinct projections from the hypothalamus to the spinal cord are discussed in the context of their possible functional significance.