The neurochemical identities of afferents to the midline thalamus were investigated in chloral hydrate-anesthetized adult Sprague-Dawley rats. The retrograde tracers, FluoroGold or cholera toxin B subunit, were centered on the paraventricular thalamic nucleus (n.Pvt), a periventricular member of the diffuse thalamocortical projection system that is reciprocally linked with visceral areas of cerebral cortex and implicated in food intake and addictive behavior. Tissues were processed with antisera raised against 5-HT, the catecholamine- synthesizing enzymes, tyrosine hydroxylase or phenylethanolamine N- methyltransferase or the cholinergic anabolic enzyme, ChAT. Serotonergic afferents principally derive from dorsal and median constituents of the mesopontine raphe. Previously unrecognized sources of catecholaminergic afferents were detected. Adrenergic afferents were traced to neurons in the C1 and C3 areas of rostral medullary reticular formation and periventricular gray, respectively, and the C2 area corresponding to the dorsal general viscerosensory field of nucleus tractus solitarii. Noradrenergic afferents arise principally from neurons in the locus ceruleus and A5 area. Dopaminergic projections to the n.Pvt derive from the A14, A13 and A11 cell groups in diencephalon. Afferents presumed to generate nitric oxide (NO) as a diffusible membrane-permeant transcellular signal were detected by processing retrogradely labeled tissues histochemically for NADPH-diaphorase, a molecule associated with nitric oxide synthase. NO in the n.Pvt is generated predominantly by noncholinergic neurons in the lateral hypothalamic area and mesopontine tegmentum. In striking contrast, extensive interactions were predicted between NO and ACh in the central medial and other loci in the nondiscriminative thalamus. We conclude that the n.Pvt is a site of interaction of NO and monoaminergic afferents derived from nuclei implicated in sensory gating, regulation of electrocortical neural activity and behavior. Taken collectively, our data predict that the labile transcellular messenger NO may enable structurally differentiated subnuclei of the diffuse thalamocortical projection system to act in concert as a functionally unified unit.