The regulatory domain of the cGMP-binding cGMP-specific 3':5'-cyclic nucleotide phosphodiesterase (PDE5) contains two homologous segments of amino acid sequence that encode allosteric cyclic nucleotide-binding sites, referred to as site a and site b, which are highly selective for cGMP over cAMP. The possibility that the state of protonation in these sites contributes to cyclic nucleotide selectivity was investigated. The binding of cGMP or cAMP was determined using saturation and competition kinetics at pH values between 5.2 and 9.5. The total cGMP binding by PDE5 was unchanged by variation in pH, but the relative affinity for cGMP versus cAMP progressively decreased as the pH was lowered. Using site-directed mutagenesis, a conserved residue, Asp-289, in site a of PDE5 has been identified as being important for cyclic nucleotide discrimination in this site. It is proposed that deprotonation of Asp-289 enhances the number and strength of bonds formed with cGMP, while concomitantly decreasing the interactions with cAMP.