The origin of neuropeptide Y (NPY) afferents to gonadotropin-releasing hormone (GnRH) neurons was investigated in male mice. Neonatal lesioning of the hypothalamic arcuate nuclei (ARC) with monosodium glutamate markedly reduced the number of NPY fibers in the preoptic area (POA) as well as the frequency of their contacts with perikarya and proximal dendrites of GnRH neurons. Dual-label immunofluorescence studies to determine the precise contribution of the ARC to the innervation of GnRH neurons by NPY axons were carried out on transgenic mice in which enhanced green fluorescent protein (GFP) was expressed under the control of the GnRH promoter (GnRH-GFP mice). The combined application of red Cy3 and blue AMCA fluorochromogenes established that 49.1 ± 7.3% of NPY axons apposed to green GnRH neurons also contained agouti-related protein (AGRP), a selective marker for NPY axons arising from the ARC. Immuno-electronmicroscopic analysis detected symmetric synapses between AGRP fibers and GnRH-immunoreactive perikarya. Additional triple-fluorescence experiments revealed the presence of dopamine--hydroxylase (DBH) immunoreactivity within 25.4 ± 3.3% of NPY afferents to GnRH neurons. This enzyme marker enabled the selective labeling of NPY pathways ascending from noradrenergic/adrenergic cell populations of the brain stem, thus defining a second important source for NPY-containing fibers regulating GnRH cells. The absence of both topographic markers (AGRP and DBH) within 26% of NPY contacts suggests that additional sources of NPY fibers to GnRH neurons exist. Future studies will address distinct functions of the two identified NPY systems in the afferent neuronal regulation of the GnRH system.