Properties of cyclic 3',5'-nucleotide phosphodiesterase in the 100,500 X g supernatant of the bovine thyroid were investigated. The enzyme activity was measured by a radioisotopic method using an anionic-exchange resin, and it was found that the activity was stimulated by Mg2+. Sephadex G-200 gel filtration separated the supernatant into an activating factor, which required the presence of Ca2+, and an enzyme form dependent on the factor. The molecular weights were estimated to be 25,000 and 130,000, respectively. There appeared to be another enzyme form of cAMP phosphodiesterase with different dependence on the activating factor as suggested by gel filtration, but this enzyme form could not be clearly separated. cGMP phosphodiesterase purified by gel filtration showed biphasic kinetic behavior in the absence of Ca2+ and the activating factor, whereas, in their presence, the Lineweaver-Burk plot gave a single Km. The activating mechanism of phosphodiesterase may play a role in the control of concentrations of intracellular cyclic 3',5'-nucleotides in the bovine thyroid.