Inhibitors of PDE3A, milrinone and cilostazol, interact with amino acids involved in cGMP binding. cGMP inhibits PDE3A. However the residues involved in cGMP binding in PDE3A are not completely categorized. We synthesized a nonhydrolyzable reactive cGMP analog, Rp-guanosine-3′,5′-cyclic-S-(4-bromo-2,3-dioxobutyl)monophosphorothioate, Rp-cGMPS-BDB with the goal to probe the cGMP site. Rp-cGMPS-BDB irreversibly inactivates PDE3A in a time-dependent fashion with KI = 43.4 ± 7.2 μM and kmax = 0.007 ± 0.0006 min−1. To identify the target site of Rp-cGMPS-BDB, nonhydrolyzable substrate and inhibitor analogs, and products were added prior to the inactivation of PDE3A by Rp-cGMPS-BDB. NAD, which is neither a substrate nor an inhibitor of PDE3A, does not protect against its inactivation by Rp-cGMPS-BDB. The effectiveness of protectants in decreasing the rate of inactivation by Rp-cGMPS-BDB is: Rp-cGMPS (Kd = 72 μM) > Sp-cGMPS (124), Sp-cAMPS (182) > GMP (1517), Rp-cAMPS (3762), AMP (4370 μM). Kinetic results from protection by Rp-cGMPS, Sp-cGMPS and Sp-cAMPS suggest that inactivation of PDE3A by Rp-cGMPS-BDB is a consequence of overlap between cAMP and cGMP binding sites but favors the cGMP site. The docking model of PDE3A-Rp-cGMPS-BDB shows that Rp-cGMPS-BDB is within the active site of PDE3A. Rp-cGMPS-BDB has proven to be an effective active site-directed affinity label for PDE3A with potential for other cGMP PDEs. Supported by PA/DE AHA grant 026439U, NIH grant HL-64943 and NSF grant MCB97-28202.