DNase I protection and gel retardation assays have identified two sequences 5' to the rat opsin gene that interact with nuclear proteins from retina but not from a number of neuronal and non-neuronal tissues. These sites, Ret 2 and Ret 3, are over 1200 base pairs apart but seem to interact with the same protein(s). Synthetic oligonucleotides corresponding to each site were able to inhibit complex formation in a gel retardation assay using an oligonucleotide corresponding to the other site. The proteins binding to the Ret 2 and Ret 3 sites co-eluted in both ion exchange and gel filtration chromatography. The protein(s) were also present at adult levels at birth, suggesting that they may represent differentiation products expressed in the proliferating retinal epithelium.