Descending projections arising from brainstem serotonergic (5HT) neurons contribute to both facilitatory and inhibitory controls of spinal cord “pain” transmission neurons. Unclear, however, are the brainstem networks that influence the output of these 5HT neurons. To address this question, here we used a novel neuroanatomical tracing method in a transgenic line of mice in which Cre recombinase is selectively expressed in 5HT neurons (ePet-Cre mice). Specifically, we injected the conditional pseudorabies virus recombinant (BA2001) that can replicate only in Cre-expressing neurons. Because BA2001 transports exclusively in a retrograde manner, we were able to reveal a subset of the neurons and circuits that are located upstream of the Cre-expressing 5HT neurons. We show that diverse brainstem regions differentially target the 5HT neurons of the dorsal raphe (DR) and the nucleus raphe magnus of the rostroventral medulla (RVM). Among these are several catecholaminergic and cholinergic cell groups, the periaqueductal gray, several brainstem reticular nuclei, and the nucleus of the solitary tract. We conclude that a brainstem 5HT network integrates somatic and visceral inputs arising from various areas of the body. We also identified a circuit that arises from projection neurons of deep spinal cord laminae V–VIII and targets the 5HT neurons of the NRM, but not of the DR. This spinoreticular pathway constitutes an anatomical substrate through which a noxious stimulus can activate 5HT neurons of the NRM and in turn could trigger descending serotonergic antinociceptive controls. J. Comp. Neurol. 514:145–160, 2009. © 2009 Wiley-Liss, Inc.