A cell-specific isozyme of calmodulin (CaM)-dependent phosphodiesterase that exhibits micromolar affinity for cAMP has been purified 900-fold from mouse testis by DEAE chromatography, gel filtration, affinity chromatography with CaM-Sepharose 4B, and isoelectric focusing. The highly purified enzyme is stimulated 5-6-fold by CaM in the presence of Ca2+ and hydrolyzes both cAMP and cGMP with anomalous substrate dependence, i.e. high and low affinity components (Km 2 and 20 microM) are observed either in the presence or absence of CaM. Each of the substrates acts as a noncompetitive inhibitor of the other, suggesting the presence of two distinct catalytic sites on the enzyme. Hydrodynamic studies suggest that the testis phosphodiesterase is an asymmetric monomer of 68-70 kDa that forms a dimer after interaction with Ca2+ and CaM; the tetrameric complex exhibits an apparent molecular size of 180 kDa. These enzymatic and biophysical properties differ in many respects from those of the brain isozyme, suggesting that they are different proteins. Nevertheless, common epitopes do exist, since the testis enzyme interacted with rabbit antibodies raised against bovine brain CaM-dependent phosphodiesterase. The major peptide of 68 kDa was strongly reactive on immunoblots, and was distinguished unambiguously from the 60-kDa species from mouse brain. A comparison of the immunoreactive fragments produced by limited proteolysis with staphylococcal V-8 protease indicated several similarities in the domains of these polypeptides. Thus, although differing in several important physical and biochemical parameters, the testis enzyme appears immunologically related to CaM-dependent phosphodiesterase from brain. On the basis of these data, we conclude that common elements of the structural genes for these isozymes have been conserved, whereas certain biological properties, including substrate specificity, have diverged substantially.