Recent evidence suggests that the two main classes of cones are not only equipped with different photopigments, but also exhibit differences in their downstream phototransduction cascade. An antibody against the γ subunit of retinal cGMP phosphodiesterase (PDE) had previously been found to label all photoreceptors in the ground squirrel (Spermophilus tridecemlineatus). This property was utilized for the cloning of a cDNA fragment encoding the corresponding polypeptide. A λ gt11 cDNA library was constructed and screened with the antibody. Positive clones were isolated, subcloned, and sequenced. Clones were used as probes in RNA blot hybridization, and the obtained sequence information was compared to other available γ-PDE sequences. Four virtually identical cDNA clones were isolated. Both nucleic acid and amino acid sequence alignment placed this gene in the same group as bovine and human cone γ-phosphodiesterase subunits. The predicted length of the translated protein was 84 amino acids, and its molecular weight 11 kD. The cDNA hybridized with an 0.6-kb transcript in retinal RNA from ground squirrel, Syrian golden hamster, and mouse, and also with a 1.4-kb transcript in the ground squirrel. We conclude that the isolated cDNA fragment encodes a γ subunit of cone cGMP-PDE. This subunit is expressed in middle-wavelength sensitive cones (the predominant photoreceptor type in this species). It remains to be determined if this subunit is expressed in short-wavelength sensitive cones as well, or if these photoreceptors are equipped with another, unique γ-PDE subunit.