Although adenosine A1 receptors mediate the inhibition of dopamine-dependent stimulation of adenylate cyclase activity in the developing chick retina, their localization and function are unknown. We have examined the localization of these receptors, and of endogenous adenosine and adenosine uptake sites at several stages of chick retinal development. A1 receptors were already localized predominantly to plexiform regions by embryonic day 12 (E12) with no gross changes at subsequent stages. Adenosine immunoreactivity was absent from retina at E8 but was detected at E12 in the ganglion cell layer, as well as cells in the inner nuclear cell layer and photoreceptors. At more advanced developmental stages the immunoreactivity was greater, but displayed similar localizations. Uptake sites labeled with [3H]nitrobenzylthioinosine (NBI) were detected even earlier using binding and autoradiographic methods. [3H]NBI binding was saturable, and Scatchard analysis demonstrated a single class of sites with a Kd of 0.91 nM and Bmax of 298 fmol/mg protein in E15 retinal membranes. The binding was displaced by unlabeled NBI and dipyridamole. NBI binding sites differentiated earlier than adenosine A1 receptors or endogenous adenosine immunoreactivity, showing a diffuse distribution at E8, but predominating in the plexiform layers of more developed retinas. The results indicate that elements of a putative purinergic system differentiate at specific localizations early in retinal development.