Rationale/Objectives: Phosphodiesterase (PDE) 5 inhibitors (e.g. Sildenafil) are selective pulmonary vasodilators in patients with pulmonary arterial hypertension (PAH). The mechanism(s) underlying this specificity remains unclear, but studies in genetically-modified animals intimate it might be dependent on natriuretic peptide bioactivity. Herein, we explored the interaction between PDE5 inhibitors and the natriuretic peptide system in vitro and in vivo, including an animal model of PH, to elucidate the (patho)physiological relationship between these two cGMP-regulating systems and potential of a combination therapy exploiting these cooperative pathways. Methods/Main Results: In vitro pharmacological studies using rat vascular tissue revealed that Sildenafil augments vasodilator responses to nitric oxide (NO) in both pulmonary and systemic conduit and resistance arteries whereas identical relaxant responses to ANP are enhanced only in pulmonary vessels. This differential activity was mirrored in vivo where Sildenafil increases the hypotensive actions of ANP in the pulmonary, but not systemic, vasculature. In an animal model of (hypoxia-induced) PH, combination treatment with Sildenafil plus the neutral endopeptidase (NEP) inhibitor Ecadotril (increases endogenous natriuretic peptide levels) acted synergistically, in a cGMP-dependent manner, to reduce many indices of disease severity (e.g. pulmonary artery pressure, right ventricular hypertrophy and pulmonary vascular re-modeling) without significantly affecting systemic blood pressure. Conclusions: These data demonstrate that PDE5 is a key regulator of cyclic GMP (cGMP)-mediated vasodilatation by ANP in the pulmonary, but not systemic, vasculature, thereby explaining the pulmonary-selectivity of PDE5 inhibitors. Furthermore, exploitation of this mechanism (i.e. PDE5 and NEP inhibition) represents a novel, orally-active combination therapy for PAH.