Many signal transduction pathways are mediated by the second messengers cGMP and cAMP, cGMP- and cAMP-dependent protein kinases (cGK and PKA), phosphodiesterases, and ion channels. To distinguish among the different cGMP effectors, inhibitors of cGK and PKA have been developed including the K-252 compound KT5823 and the isoquinolinesulfonamide H89. KT5823, an in vitro inhibitor of cGK, has also been used in numerous studies with intact cells to implicate or rule out the involvement of this protein kinase in a given cellular response. However, the efficacy and specificity of KT5823 as cGK inhibitor in intact cells or tissues have never been demonstrated. Here, we analyzed the effects of both KT5823 and H89 on cyclic-nucleotide-mediated phosphorylation of vasodilator-stimulated phosphoprotein (VASP) in intact human platelets and rat mesangial cells. These two cell types both express high levels of cGK. KT5823 inhibited purified cGK. However, with both intact human platelets and rat mesangial cells, KT5823 failed to inhibit cGK-mediated serine 157 and serine 239 phosphorylation of VASP induced by nitric oxide, atrial natriuretic peptide, or the membrane-permeant cGMP analog, 8-pCPT-cGMP. KT5823 enhanced 8-pCPT-cGMP-stimulated VASP phosphorylation in platelets and did not inhibit forskolin-stimulated VASP phosphorylation in either platelets or mesangial cells. In contrast H89, an inhibitor of both PKA and cGK, clearly inhibited 8-pCPT-cGMP and forskolin-stimulated VASP phosphorylation in the two cell types. The data indicate that KT5823 inhibits purified cGK but does not affect a cGK-mediated response in the two different cell types expressing cGK I. These observations indicate that data that interpret the effects of KT5823 in intact cells as the major or only criteria supporting the involvement of cGK clearly need to be reconsidered.