cGMP affects vascular tone by multiple mechanisms including inhibition of the Rho/Rho kinase mediated Ca²⁺ sensitization, a process identified as Ca²⁺ desensitization. Ca²⁺ desensitization is mediated probably by both cGMP- and cAMP-dependent protein kinases (cGKI and PKA). We investigate to which extent Ca²⁺-desenzitisation is initiated by cGKI and PKA. cGMP/cAMP-induced relaxation was studied at constant [Ca²⁺] in permeabilized aortae from wild type and cGKI-deficient mice. [Ca²⁺] increased aortic tone in the absence and presence of 50 µM GTPS with EC₅₀'s of 160 and 30 nM, respectively. In the absence of GTPS, the EC50 for [Ca²⁺] was shifted rightwards from 0.16 µM to 0.43 and 0.82 by 1 and 300 µM 8-Br-cGMP, and to 8 µM by 10 µM Y-27632. Contractions induced by 300 nM [Ca²⁺] were relaxed by 8-Br-cGMP with an EC₅₀ of 2.6 µM. Surprisingly, [Ca²⁺]-induced contractions were also relaxed by 8-Br-cGMP in aortae from cGKI^-/- mice (EC50 of 19 µM). Western blot analysis of the vasodilator-stimulated phosphoprotein indicated "cross"-activation of PKA by 1 mM 8-Br-cGMP in aortic smooth muscle cells from cGKI^-/- mice. Indeed, the PKA inhibitor peptide (PKI 5-24) completely abolished the relaxant effect of 8-Br-cGMP in muscles from cGKI^-/- mice and to 65 % in wild type aortae. The thromboxane-analogue U46619 induced contraction at constant [Ca²⁺] which was only partially relaxed by 8-Br-cGMP, but completely by Y-27632. The effect of 8-Br-cGMP on U46619-induced contraction was attenuated by PKI 5-24. These results show that cGKI has only a small inhibitory effect on Ca²⁺ sensitization in murine aorta.