The reticular nucleus (RT) of the thalamus, a thin sheet of GABAergic neurons located between the external medullary lamina and the internal capsule of the thalamus, has functionally distinct afferent and efferent connections with thalamic nuclei, the neocortex, the basal forebrain and the brainstem. RT is critically positioned to rhythmically pace thalamocortical networks leading to the generation of spindle activity during the early phases of sleep and during absence (spike-wave) seizures. Serotonin, acting on 5-HT1A receptors on parvalbumin-containing cells of RT, has been implicated in this rhythmicity. However, the precise source(s) of 5-HT afferents to the RT remains to be determined. In the present study, we injected the retrograde tracer, Fluorogold, into dorsal and ventral regions of RT to determine the origins of raphe input to RT. We further characterized the distribution of 5-HT fibers to RT by using immunohistochemistry for 5-HT and for the 5HT transporter (SERT) detection. Finally, we described the presence of the two major postsynaptic 5-HT receptors in RT, 5-HT1A and 5-HT2A receptors. Our results show that the dorsal raphe nucleus and the supralemniscal nucleus (B9) of the midbrain are the principal sources of raphe projections to RT. In addition, serotonergic fibers (5-HT and SERT positive) were richly distributed throughout RT, and 5-HT1A and 5-HT2A receptors were highly expressed on RT neurons and dendrites. These findings suggest a significant 5-HT modulatory influence on GABAergic neurons of RT in the control of rhythmical (or spindle) activity in thalamocortical systems directly associated with sleep and possibly with absence seizures. Synapse, 2011. © 2011 Wiley-Liss, Inc.