Therapies for pulmonary hypertension include the phosphodiesterase (PDE) 5 inhibitor sildenafil and prostacyclin, which raise cGMP and cAMP respectively. Pulmonary hypertension is associated with a Rho kinase-mediated increase in Ca²⁺ sensitivity in pulmonary arteries, and it has been suggested that cGMP and cAMP may suppress this pathway. We studied this hypothesis in intrapulmonary arteries (IPA)of the rat, mounted on a myograph; experiments were performed on intact or α-toxin permeabilised IPA. In intact IPA constricted with 10 μM PGF2α, 8-Br-cGMP (cGMP analogue) elicited relaxation with an EC₅₀ of 20±13μM and maximum relaxation of 102±3% (n=6). In permeabilised arteries at pCa 6.7 the potency of 8-Br-cGMP was much greater, with an EC₅₀ of 18±4nM and maximum relaxation of 64±5% (n=5). 8-Br-cGMP (100nM) also caused a rightward shift of the Ca²⁺ dose-response curve, with an increase in EC₅₀ from 165 ± 5 nM to 316 ± 14 nM (n=44; p<0.05), indicating calcium desensitisation. Complete inhibition of Rho kinase with Y-27632 (10µM) did not prevent this shift (EC₅₀: Y-27632: 205 ± 26 nM, n=8; Y-27632+8-bromo-cGMP: 306 ± 26 nM (n=6). This suggests that cGMP does not act via Rho kinase. Interestingly, 8-Br-cGMP-induced relaxation of permeabilised IPA was suppressed not only by the cGMP antagonist Rp-8Br-cGMP (25µM) (control: 77 ± 3%; Rp-8Br-cGMP: 44 ± 3%, n=4-8, p<0.01), but also by the cAMP antagonist Rp-8Br-cAMP (25µM) (43 ± 5%; n=4; p<0.01); there was no increase when these were applied together (43 ± 9%, n=3), suggesting that cGMP and cAMP may be acting sequentially. cGMP inhibits cAMP-selective PDE 3, and the PDE 3 inhibitor milrinone proved to be a powerful vasorelaxant in intact IPA (EC₅₀ 21 ± 7 μM, maximum 100 ± 13% , n=4). To determine whether cGMP elevates intracellular [cAMP], we used an ELISA and cultured smooth muscle cells derived from rat IPA. 8-Br-GMP (100µM) increased cAMP from 25 ± 3 pmol/mg protein to 48 ± 6 pmol/mg protein (n=16, p<0.001), whereas 10μM milrinone increased it to 43 ± 6 pmol/mg protein (n=10, p<0.01). As 10μM milrinone, which acts via cAMP, causes ~50% relaxation of intact IPA, the elevation in cAMP caused by 8-bromo-cGMP is clearly sufficient to account for a significant component of vasorelaxation. This suggests that cGMP-mediated Ca²⁺ desensitisation may involve cAMP and PKA; consistent with this, the protein kinase A inhibitor H-89 (10μM) abolished the effects of 8-Br-cGMP on the Ca²⁺ dose-response curve, although H-89 itself caused a significant shift in response (EC₅₀: H-89: 533 ± 81 nM; H-89 + 8-Br-cGMP: 550 ± 57 nM (n=4). In conclusion, we show that cGMP-induced Ca²⁺ desensitisation of IPA is independent of Rho kinase, but that a significant component of relaxation may be related to a cAMP and PKA-dependent pathway.