Brainstem monoamines have long been considered to play a role in regulating the activity of GnRH neurons, although their neuroanatomical relationship with these cells has remained unclear. Using a Cre-dependent pseudorabies virus (Ba2001) technique that permits retrograde tracing selectively from GnRH neurons in the mouse, we have examined the organization of brainstem inputs to rostral preoptic area (rPOA) GnRH neurons. Two days after injection of Ba2001 into the rPOA of adult female GnRH-Cre transgenic mice, five to nine GnRH neurons located immediately adjacent to the injection site were found to express green fluorescent protein (GFP), the marker of virus infection, with no GFP expression anywhere else in the brain. In mice killed 24 h later (3 d after injection), GFP-expressing cells were identified (in order of density) in the raphe nuclei, periaqueductal grey, locus coeruleus, nucleus tractus solitarius, and area postrema. This time course is compatible with these neurons representing primary afferent inputs to the GnRH neurons. Four and 6 d after Ba2001 injection, GFP-expressing cells were found in additional brain regions. Dual-label immunofluorescence experiments in 3-d postinjection mice demonstrated that 100% of GFP-expressing neurons in the raphe were positive for tryptophan hydroxylase, whereas 100% and approximately 50% of GFP neurons in the locus coeruleus and nucleus tractus solitarius, respectively, expressed tyrosine hydroxylase. These observations demonstrate that rPOA GnRH neurons receive direct projections from brainstem A2 and A6 noradrenergic neurons and that, surprisingly, the largest afferent input from the brainstem originates from raphe serotonin neurons in the mouse.