Publisher Summary This chapter gives an overview on cyclic GMP-specific cGMP-binding phosphodiesterase (PDE5), a class I mammalian PDE with a catalytic site that specifically hydrolyzes the phosphodiester bond of cGMP and an allosteric site that binds cGMP with high specificity. The single human PDE5 gene is located on chromosome 4q26, contains 23 exons, and encodes mRNA for three variants, PDE5A1, -A2, and -A3. The protein products, 100 kDa, 95 kDa, and 95 kDa, respectively, share similar properties and have different amino-termini, a result of alternative mRNA splicing. Phosphorylation of Ser-102 (human PDE5) by PKG or PKA is enhanced by cGMP binding to the regulatory region or ligand (cGMP or PDE5 inhibitors) occupation of the catalytic site. PDE5 is phosphorylated in intact cells in response to stimuli that elevate cGMP, but elevation of cAMP does not mimic this. Cloning of PDE5 cDNA led to identification of two conserved Zn2+ binding motifs in catalytic domains of all mammalian PDEs. Zinc that binds to PDE5 with high stoichiometry is effective at a far lower concentration than either Mn2+ or Mg2+ in supporting catalytic activity. An allosteric site in unphosphorylated PDE5 binds cGMP with a KD ∼0.2-1.5 μM; affinity varies depending on conditions. Cyclic GMP in the anti conformation binds to GAF-A, which is composed of ∼110 residues. A recombinant protein containing only GAF-A binds cGMP with high affinity, and cGMP binding causes a large conformational change. GAF-A also provides high-affinity dimerization contacts between PDE5 monomers. GAF-B provides high-affinity dimerization contacts between PDE5 monomers and reduces affinity of the GAF-A subdomain for cGMP.