We investigated the InsP6 binding proteins in bovine retinal membranes and rod outer segments (ROS) by radioligand binding assay and western blotting. The relative affinity of InsP6 for the binding protein was determined by competitive binding of [3H]-InsP6 with increasing concentrations of the unlabeled InsP6 or other isomers. InsP6 specifically binds to both bovine retinal membranes and ROS; maximum binding was achieved after one-hour incubation at 4 degrees C and was unchanged up to 2 h. Tris-HCl or acetate buffer was equally suitable for the binding assay over a broad range of pH, although specific binding was slightly increased at acidic pH. The order of potencies of displacement was InsP6 > Ins(1,3,4,5,6)P5 > Ins(1,3,4,6)P4 = Ins(1,3,4,5)P4, whereas Ins(1,4,5)P3, Ins(1,4)P2, Ins(4,5)P2, and Ins(1)P were not effective displacers. Scatchard analyses of the binding data were consistent with an equilibrium dissociation constant (Kd) of 2.5 +/- 0.2 microM and maximal binding capacity (Bmax) of 123.7 +/- 25.0 pmol/mg at pH 7.4. Western blotting was used to detect whether AP-2 (an InsP6 binding protein) is present in the retina. Immunoreactivity to AP-2 alpha and beta subunits was found in retinal membranes and ROS. Thus, bovine retinal membranes and ROS contain membrane-associated InsP6 binding protein(s) which is distinct from proteins that bind InsP5, InsP4, or InsP3.