The neural substrate of the pupillary light reflex in the pigeon was investigated using anatomical, stimulation, and lesion techniques. In birds, as in mammals, the sphincter pupillae muscle (which constricts the iris) is innervated by cells in the ciliary ganglion (Pilar and Tuttle, '82). These cells are in turn innervated by cells in the Edinger-Westphal nucleus (EW) (Cowan and Wenger, '68; Narayanan and Narayanan, '76; Lyman and Mugnaini, '80). The efferent link of the pupillary light reflex must therefore involve cells in EW. To study the central course of this reflex pathway, injections of horseradish peroxidase (HRP) were placed in EW. These injections labeled cells in a number of regions including a contralateral pretectal nucleus, area pretectalis (AP). Only a limited number of cells in AP project to EW. Injections of tritiated amino acids into AP labeled a discrete region of the contralateral EW. This projection is confined to a dorsolateral region of caudal EW and overlies the somata of approximately 100 cells. Tritiated proline was injected into the eye, and the results confirmed an earlier report (Reperant, '73) that AP receives retinal input from the contralateral eye. Immunohistochemical studies demonstrated fibers in AP that stained positively for substance-P-like, enkephalin-like and tyrosine-hydroxylase-like immunoreactivity. Injections of HRP were placed in AP to examine the retinal ganglion cells mediating the reflex. Cells with an average diameter of approximately 14 microns (5-25 microns range) were labeled and averaged approximately 6 microns greater in diameter than the retinal ganglion cells (mean = 7.3 microns) labeled by an optic chiasm injection. The cells labeled by AP injections were distributed unevenly throughout the retina with a higher concentration in the central and temporal retina and a paucity in the red field and fovea. Our results demonstrate that AP receives input from a distinct subpopulation of large retinal ganglion cells that comprises a very small percentage of the total population of retinal ganglion cells. Unilateral lesions of AP abolished the pupillary light reflex in the eye contralateral to the lesion; stimulation of AP elicited pupilloconstriction in the eye contralateral to the stimulation site. These results delineate the central course of the pupillary light reflex pathway in the pigeon and identify the retinal ganglion cells that subserve this reflex. They show that, at every point in the pathway, only a few cells mediate this simple reflex.(ABSTRACT TRUNCATED AT 400 WORDS)