cGMP is the second messenger for visual excitation in vertebrate rod photoreceptors. However, no direct correlation has been observed between the measured total cGMP concentration in the rod outer segment and the electrical response of these cells to photic stimulation. To address this discrepancy, we have quantitated the number and affinities of cGMP binding sites in the rod outer segment to determine the cytoplasmic free cGMP concentration that is involved in visual transduction. We identified two distinct classes of cGMP binding sites in amphibian rod outer segments: 1) high affinity binding sites with a KD1 = 60 nM and a site density of 30 microM, and 2) moderate affinity binding sites with a KD2 = 6.6 microM and a site density of 78 microM. These two classes of binding sites are calculated to bind 94% of the total cellular cGMP, thereby lowering the cytoplasmic cGMP concentration to 3.5 microM in dark-adapted rod outer segments. This value is consistent with predictions of the cytoplasmic cGMP concentration based on activation of the cGMP-gated ion channel of rod photoreceptors. The kinetics of cGMP dissociation from high affinity binding sites indicate that this class of sites would dissociate its bound cGMP too slowly to participate in visual excitation and recovery to flash illumination. This binding of cGMP to intracellular binding sites provides a non-enzymatic mechanism by which photoreceptor cells regulate the concentration and restrict the diffusion of this second messenger during visual transduction.