Cyclic AMP and cyclic GMP phosphodiesterases (3':5'-cyclic-nucleotide 5'-nucleotidohydrolase EC 3.1.4.17) were found in the sera of human, dog, rabbit and rat. The formed elements of blood were not present in sera and thus not the source of the phosphodiesterase. More rapid hydrolysis of cyclic GMP than cyclic AMP is observed in the sera of these four species when 0.4 muM of cyclic AMP or cyclic GMP is used as the substrate. Protein activator of the phosphodiesterase is not detectable in the sera of these four species. Serum cyclic AMP and cyclic GMP phosphodiesterase activities are not stimulated by protein activator prepared from bovine brain. The serum phosphodiesterases of these four species are purified through Sepharose 6B column chromatography. Cyclic AMP phosphodiesterase are found in a broad area corresponding to molecular weights ranging from approximately 150 000 to 340 000 with 2 to 3 peaks in all animals tested. Cyclic GMP hosphodiesterase is found in a single area corresponding to molecular weights of 230 000 (rabbit and rat) and 270 000 (human and dog). Serum cyclic AMP and cyclic GMP hosphodiesterase activities of these four species have pH optimum of 7.5-8.5. Optimal concentration of Mg-2+ is about 5 mM for cyclic GMP phosphodiesterase activities of these four species as well as for cyclic AMP phosphodiesterase activities except rabbit. Rabbit serum cyclic AMP phosphodiesterase requires higher concentration of Mg-2+ (50 mM) for its optimal activity. The double reciprocal plots are non-linear for cyclic AMP phosphodiesterases of all animals and cyclic GMP phosphodiesterases of human, dog and rat. Rabbit cyclic GMP phosphodiesterase exhibits a linear reciprocal plot. Cyclic GMP is inhibitor of serum cyclic AM phosphodiesterase. Rabbit enzyme was most effectively inhibited by cyclic GMP.