A significant component of vascular smooth muscle relaxation caused by agents that raise intracellular [cyclic GMP] (cGMP) has been proposed to depend on [Ca2+]i desensitisation involving an interference with rho kinase-mediated inhibition of myosin phosphatase. However, the role of [Ca2+]i desensitisation in response to cGMP is not well understood in pulmonary artery smooth muscle. Using small vessel myography, we characterised the vasorelaxation evoked by the cell permeable cGMP analogue 8-Br-cGMP. 100 nM 8-Br-cGMP-induced relaxation in α-toxin permeabilised rat intra-pulmonary arteries (IPA) (at pCa 6.7, EC50=18±4 nM, Vmax=63±4 %). This relaxation was unaffected by the PKG inhibitor, KT5823 (5µM; 62.9±4%, n=16 vs 65±7%, n=21 in controls), by the rho kinase inhibitor Y-27632, (10µM; 73±13%, n=6 as compared to 76±9%, n=7 in controls), or by the protein kinase C inhibitor Ro-31-8220 (3µM; 89±5%, n=3 vs 80±4, n=4 in controls). To investigate the involvement of protein kinase A in 8-Br-cGMP-mediated relaxation of permeabilised IPA, we applied PKI (14-22). 20 µM of PKI had no effect on 100 nM 8-Br-cGMP-mediated relaxation in permeabilised IPA (controls 68±6%, n=4, pre-treatment with PKI 62±7%, n=4). PKI also did not reduce the relaxation to 100 nM 8-Br-cAMP in permeabilised IPA (control 89±4%, n=4, PKI 84±1%, n=4). Relaxation caused by 100 nM 8-Br-cGMP in permeabilised IPA (80±5%, n=5) was inhibited by the cGMP antagonist Rp-8Br-cGMP (25µM) (to 45±9%, n=5) and the cAMP antagonist Rp-8Br-cAMP (25µM) (to 54±7%, n=5). Unexpectedly, in IPA both antagonists themselves caused relaxation of the contraction produced by pCa 6.8 (relaxation of 39±5% for Rp-8Br-cGMP, n=4 and 32±4% for 8-Br-cAMP, n=4). Conversely, when added to permeabilised mesenteric arteries pre-constricted with pCa 5.8, these antagonists caused a variable constriction. Relaxation to 100 nM 8-Br-cGMP in permeabilized mesenteric arteries (42±8%, n=7) was significantly inhibited by 25µM Rp-8Br-cGMP to 20±6% (n=6) but not by 25µM Rp-8Br-cAMP (to 36±7%, n=6). Our data indicate that that in IPA cGMP causes Ca2+ desensitisation cGMP through a Rho-and PKC-independent pathway. Moreover, the effects of the pharmacological blockers of cGMP and cAMP-dependent pathways which we tested suggest that cyclic nucleotides act differently in systemic vs pulmonary arteries, and may be able to cause Ca2+ desensitisation via pathways independent of PKA and/or PKG in the latter.