INAD is a scaffolding protein containing five PSD95/dlg/zonular occludens-1 (PDZ) domains that tether NORPA (phospholipase Cbeta(4)), the TRP calcium channel, and eye-PKC in Drosophila photoreceptors. We previously showed that eye-PKC interacted with the second PDZ domain (PDZ2) of INAD. Sequence comparison with a prototypical type I PDZ domain predicts that PDZ2 is the best candidate among the five PDZ domains to recognize eye-PKC that contains a type I PDZ ligand, Ile-Thr-Ile-Ile, at its carboxyl terminus. Replacement of Ile(-3) in eye-PKC with charged residues resulted in a drastic reduction of the PDZ2 interaction. Substitution of a conserved His with Arg at the second alpha-helix of PDZ2 led to a reduced binding; however, a Leu replacement resulted in an enhanced eye-PKC association. We isolated and sequenced the InaD gene. The coding sequence of InaD contains nine exons spanning 3 kilobases. Translation of coding sequences from three wild-type alleles revealed three SNPs affecting residues, 282, 319, and 333 of INAD. These polymorphisms are localized in PDZ2. Interestingly, we found two of three PDZ2 variants displayed a greater affinity for eye-PKC. In summary, we evaluated the molecular basis of the eye-PKC and PDZ2 association by mutational analysis and concluded that PDZ2 of INAD is a type I domain important for the eye-PKC interaction.