The avian optic tectum is composed of at least 15 separate laminae that are distinguishable on the basis of their morphological features and patterns of afferent and efferent connectivity. Layer 5b, a major retinorecipient layer, exhibits dense, dust-like, neuropeptide Y-positive (NPY+) immunoreactive labeling, whereas sparse, larger caliber NPY+ fibers are found in laminae 4 and 7. Anterograde and retrograde labeling techniques, immunohistochemistry, and retinal lesion studies were used to determine the source of this tectal NPY+ labeling. NPY+ was not detectable in cells of the optic tectum or in retinal ganglion cells, and retinal ablation did not diminish the abundance of tectal NPY+ fibers. Neurons of two nuclei previously shown to be sources of tectal input, the nucleus pretectalis (PT) and the intergeniculate leaflet (IGL; Brecha, 1978), were found to be NPY+. Unilateral injection of retrograde tracers into the tectum resulted in bilateral labeling of neurons within PT, and injections of anterograde tracer into PT confirmed that this nucleus projected bilaterally to layer 5b of the optic tectum. Unilateral lesions of PT nearly eliminated NPY+ fibers in the ipsilateral layer 5b and significantly reduced them in the contralateral layer 5b. Bilateral lesions of PT eliminated NPY+ fibers bilaterally in layer 5b. However, these PT lesions had little effect on the NPY+ fibers in layers 4 and 7. Combined retrograde and immunohistochemical studies showed that NPY+ neurons of the IGL project to the optic tectum, and anterograde studies demonstrated that IGL projects to layers 4 and 7. The NPY+ projection to laminae 5b from PT is one of many inputs, which include cholinergic afferents from the nucleus isthmi parvicellularis, terminals from retinal ganglion cells, and dendrites of layer 13 neurons (Karten et al., 1993). The NPY+ input to layer 5b may modulate visual information flow from retinal input to various tectal neurons, including those in layer 13.