Perinatal adverse events such as limitation of nutrients or oxygen supply are associated with the occurrence of diseases in adulthood, like cardiovascular diseases and diabetes. We investigated long-term effects of perinatal hypoxia on the lung circulation, with particular attention to the nitric oxide/cGMP pathway. Mice were placed under hypoxia in utero 5 days before delivery and for 5 days after birth. Pups were then bred in normoxia until adulthood. Adults born in hypoxia displayed altered regulation of pulmonary vascular tone with higher right ventricular pressure in normoxia and increased sensitivity to acute hypoxia as compared to controls. Perinatal hypoxia dramatically decreased endothelium-dependent relaxation induced by acetylcholine in adult pulmonary arteries (PA) but did not influence nitric oxide-mediated endothelium-independent relaxation. The muscarinic receptor M3 was implicated in the relaxing action of acetylcholine and the muscarinic receptor M1 (M1AChR) in its vasoconstrictive effects. Pirenzepine or telenzepine, two preferential inhibitors of M1AChR, abolished the adverse effects of perinatal hypoxia on acetylcholine-induced relaxation. M1AChR mRNA expression was increased in lungs and PA of mice born in hypoxia. The phosphodiesterase 1 (PDE1) inhibitor vinpocetine also reversed the decrease in acetylcholine-induced relaxation following perinatal hypoxia, suggesting that M1AChR-mediated alteration of acetylcholine-induced relaxation is due to activation of the calcium-dependent PDE1. Therefore, perinatal hypoxia leads to altered pulmonary circulation in adulthood with vascular dysfunction characterized by impaired endothelium-dependent relaxation and M1AChR plays a predominant role. This raises the possibility that muscarinic receptors could be key determinants in pulmonary vascular diseases in relation to "perinatal imprinting".