Cyclic nucleotide phosphodiesterases (PDEs) regulate intracellular levels of cAMP and cGMP by hydrolyzing them to their corresponding 5' monophosphates. We report here the cloning and characterization of a novel cAMP-specific PDE from mouse testis. This unique phosphodiesterase contains a catalytic domain that overall shares <40% sequence identity to the catalytic domain of all other known PDEs. Based on this limited homology, this new PDE clearly represents a previously unknown PDE gene family designated as PDE8. The cDNA for PDE8 is 3,678 nucleotides in length and is predicted to encode an 823 amino acid enzyme. The cDNA includes a full ORF as it contains an in-frame stop codon before the start methionine. PDE8 is specific for the hydrolysis of cAMP and has a Km of 0.15 microM. Most common PDE inhibitors are ineffective antagonists of PDE8, including the nonspecific PDE inhibitor 3-isobutyl-1-methylxanthine. Dipyridamole, however, an inhibitor that is generally considered to be relatively specific for the cGMP selective PDEs, does inhibit PDE8 with an IC50 of 4.5 microM. Tissue distribution studies of 22 different mouse tissues indicates that PDE8 has highest expression in testis, followed by eye, liver, skeletal muscle, heart, 7-day embryo, kidney, ovary, and brain in decreasing order. In situ hybridizations in testis, the tissue of highest expression, shows that PDE8 is expressed in the seminiferous epithelium in a stage-specific manner. Highest levels of expression are seen in stages 7-12, with little or no expression in stages 1-6.