Retinoid signaling has been implicated as an important regulator of retinal development and differentiation. We have used state of the art high-pressure liquid chromatography to identify and quantitate biologically active retinoids, immunohistochemistry to localize the retinoic acid synthetic enzyme retinaldehyde dehydrogenase 2 (RALDH2), and nucleic acid assays to quantitate and localize retinoid receptor gene transcripts in the developing eye and retina of the chicken. Our results demonstrate spatial distinctions in retinoid synthesis and signaling that may be related to laminar differentiation in the developing retina. Retinoic acids (RAs) and their precursor retinols (ROHs) are the predominant retinoids in the developing eye. All-trans-RA and all-trans-3,4-didehydro-RA are present in the neuroepithelium in approximately equal amounts from early stages of neurogenesis until shortly before hatching. The retinoid X receptor (RXR) ligand 9-cis-RA is undetectable at all stages; if present, it cannot exceed a small percentage of the total RA content. RAs are not detected in the pigment epithelium. All-trans-ROH is present in the neuroepithelium and pigment epithelium, whereas all-trans-3,4-didehydro-ROH is detected only in the pigment epithelium and/or the choroid and sclera. RALDH2 immunoreactivity is intense in the choroid, low or absent in the pigment epithelium, and moderate in the neuroepithelium, where it is highest in the outer layers. Transcripts of all five chicken retinoid receptor genes are present in the neural retina and eye throughout development. During the period of neurogenesis, at least three of the receptors (RAR gamma, RXR gamma, RXRalpha), exhibit dynamic patterns of differential localization within the depths of the neural retina.