DNase I protection and gel retardation assays have identified a sequence 5' to the transcription start site of the rat opsin gene that interacts with nuclear proteins from mammalian retinas but not from a variety of other neural and non-neural tissues. Following sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transfer to nitrocellulose the protein(s) responsible for this binding were identified with an oligonucleotide probe and were found to migrate with an apparent molecular size of 40 kilodaltons. The binding complex eluted from fast protein liquid chromatography gel filtration as a peak centered at 100 kilodaltons, suggesting the presence of more than one subunit. Binding activity could be detected in postnatal day 1 retinal extracts and increased over the next 2 weeks of development, a time course coincident with opsin gene expression and maturation of rod photoreceptors. Synthetic oligonucleotides with altered sequences showed that the binding was dependent upon residues in a CTAAT motif and was facilitated by surrounding GGCCCC sequences. The specificity of the binding interaction was measured by inhibition of complex formation in a gel retardation assay. The unaltered sequence was over 2 orders of magnitude more effective at inhibiting complex formation than either an unrelated DNA sequence or a concensus sequence corresponding to a known CCAAT box binding protein NF1.