The NO/cGMP-dependent protein kinase I (cGKI) pathway affects vascular tone by multiple mechanisms including inhibition of Ca2+-sensitization. The contribution of this mechanism to cGMP-induced relaxation was studied in permeabilized aorta from mice. The EC50 of [Ca2+]-induced contraction was shifted from 160 nM to 30 nM by 50 mM GTPgS. In the absence of GTPgS, the EC50 of [Ca2+]-induced contraction was shifted from 0.16 mM to 0.43 mM and 0.82 mM by 1 and 300 mM 8-Br-cGMP, and to 8 mM by 10 mM Y-27632. In wild type aortas, 8-Br-cGMP relaxed contractions induced by 300 nM [Ca2+] with an EC50 of 2.6 mM. Surprisingly, [Ca2+]-induced contractions (300 nM) were also relaxed by 8-Br-cGMP in cGKI-deficient (cGKI-/-) mice (EC50 of 19 mM). Preincubation of permeabilized aortic strips with the protein kinase A inhibitor peptide (5–24) completely abolished the relaxant effect of 8-Br-cGMP in cGKI-/--aortas. In muscles of wild type mice, the inhibitor reduced relaxation of 8-Br-cGMP by about 60 %. Western blot analysis of the vasodilator-stimulated phosphoprotein (VASP) supported 'cross'-activation of protein kinase A by 8-Br-cGMP. These results demonstrate, that (i) protein kinase A is involved in relaxation induced by 8-Br-cGMP and (ii) cGKI plays only a small role in relaxation after Ca2+-sensitization in murine aorta.