To determine the presence of cone or rod cyclic GMP phosphodiesterase (EC 3.1.4.17) in the mammalian pineal, extracts from adult rat and bovine pineals were injected onto a Mono Q anion-exchange HPLC column and eluted with an NaCl linear gradient. Fractions were immunoadsorbed with monoclonal antibodies specific to rod and cone phosphodiesterases (ROS-1) and to calmodulin-phosphodiesterase complexes (ACC). Profiles were assayed with 10 mumol/L [3H]cyclic GMP in the presence of calcium-calmodulin, histone, or trypsin. Rat and bovine pineals displayed a single peak of activity recognized by ROS-1, which corresponded to the activity of the cone but not to the rod in bovine retina. ROS-1 immunoadsorbed approximately 80% of the activity in the 60-day-old rat pineal but only 26% of the activity in bovine pineal. ACC immunoadsorbed the remaining activity in both species. Western blot analysis of rat pineal extracts revealed three polypeptides of approximately 87, 15, and 10 kDa when probed with a rod/cone phosphodiesterase-specific antiserum. The specific activity of the cone-like phosphodiesterase in 10-day-old rat pineals was twice that of this isozyme in the bovine retina and 150 times that in the bovine pineal. The specific activity of phosphodiesterase in rat pineals decreased with age. We conclude that an enzyme with biochemical and antigenic characteristics similar to cone, but distinct from rod phosphodiesterase, is present in bovine and rat pineals.