Cone cyclic nucleotide-gated (CNG) channels, composed of CNGA3 and CNGB3 subunits, play a central role in color vision. Along with other genes, mutations in CNGB3 are associated with achromatopsia, an inherited autosomal recessive disorder that results in loss of color vision and cone function. Two independent canine colonies were bred with mutations in CNGB3; the affected dogs lacked cone ERG function. One mutant colony has a missense mutation at residue 262 with Asp replaced by Asn, denoted CNGB3m/m, and the other colony has a deletion resulting in loss of the CNGB3 gene, denoted CNGB3-/- . We examined expression of cone CNG channels in the retinas of carrier (CNGB3m/+ or CNGB3-/+) and affected dogs using immunohistochemistry and immunoblotting with an anti-canine CNGA3 antibody, qRT-PCR and in-situ hybridization. qRT-PCR shows that missense-CNGB3 mRNA is expressed in retinal extracts of CNGB3m/m dogs but not in CNGB3-/- dogs. Of Interest, retinas from all affected dogs show complete loss of CNGA3 immunoreactivity in the outer segments despite evidence that both the CNGA3 mRNA and the protein are expressed in normal and affected retinas. Affected dogs were treated with rAAV-mediated gene replacement therapy with the human CNGB3 transgene using a human red cone opsin promoter. A single subretinal injection in one eye was sufficient to restore cone ERG function in a majority of treated dogs. Examination of CNGA3-immunoreactivity in the retinas of treated dogs showed that the human CNGB3 transgene directed CNGA3 expression to the cone outer segment. Future studies will monitor CNG channel subunit composition directly to address whether the Asp262Asn mutation can participate in CNG channel formation.