1. Homomeric cyclic nucleotide-gated (CNG) channels composed of alpha2 subunits from bovine cone photoreceptors were heterologously expressed in the human embryonic kidney (HEK) 293 cell line. Modulation of cGMP sensitivity by protein kinase C (PKC)-mediated phosphorylation and by binding of calmodulin (CaM) was investigated in inside-out patches. 2. A peptide encompassing the putative CaM-binding site within the N-terminus of the channel protein binds Ca(2+)-CaM with high affinity, yet the ligand sensitivity of alpha2 channels is not modulated by CaM. 3. PKC-mediated phosphorylation increased the activation constant (K(1/2)) for cGMP from 19 to 56 microM and decreased the Hill coefficient (from 2.5 to 1.5). The change in ligand sensitivity involves phosphorylation of the serine residues S577 and S579 in the cGMP-binding domain. The increase in K(1/2) was completely abolished in mutant channels in which the two serine residues were replaced by alanine. 4. An antibody specific for the delta isoform of PKC strongly labels the cone outer segments. 5. Modulation of cGMP affinity of bovine alpha2 CNG channels by phosphorylation could play a role in the regulation of photoreceptor sensitivity.