Rods and cones have analogous phototransduction components and cycles, but differ from each other in their physiological response to light. Differences between the affinities of rod and cone phosphodiesterase (PDE) catalytic subunits for their respective inhibitory subunits could potentially contribute to these physiological differences. To test this idea, we expressed both the 13 kDa PDE subunit, unique to a subset of bovine retinal cones [(1990) J. Biol. Chem. 265, 11259-11264], and the rod PDE 11 kDa inhibitory subunit in E. coli, purified them, and compared their abilities to inhibit rod and cone PDE catalytic subunits. Rod PDE has similar Ki values (approximately 80 pM) for both the rod and cone recombinant inhibitory subunits. Activated cone PDE has Ki values of 200 pM for the cone 13 kDa subunit and 600 pM for rod PDE gamma.