A cDNA clone corresponding to a novel cGMP phosphodiesterase inhibitory subunit (PDE gamma) was isolated from a bovine retinal cDNA library using a rod PDE gamma cDNA as a probe under conditions of reduced stringency. This new clone encodes an 83-amino acid protein approximately 80% identical to rod PDE gamma. The transcript for this protein is retina specific and 940 base pairs in length. Southern blots of bovine genomic DNA show that transcripts encoding rod gamma and this new PDE gamma are derived from different genes. Polyclonal antibodies raised against peptides corresponding to the unique amino termini of the rod PDE gamma and this new clone label rod outer segments and a subset of cone outer segments, respectively, on bovine retinal sections. This subset of cones is also recognized by antibodies that label human blue cones. On Western blots, antibodies against this new cone protein specifically recognize a 13-kDa cone PDE inhibitor.