Recent evidence suggests that endocannabinoids acting via cannabinoid CB₁ receptors may modulate vascular responses of various vasoconstrictors in the rodent systemic vasculature. The aim of the study was to investigate whether endocannabinoids modulate the contractile responses evoked by a thromboxane A₂ analog (U46619), angiotensin II (Ang II), serotonin (5-HT) and phenylephrine which stimulate distinct G_q/11-protein coupled receptors (TP, AT1, 5-HT2 and α1-adrenergic) in isolated endothelium-intact human (hPAs) and rat pulmonary arteries (rPAs). The CB₁ receptor antagonist AM251 (1 μM) and diacylglycerol lipase (2-arachidonoylglycerol synthesis enzyme) inhibitor RHC80267 (40 μM) enhanced contractions induced by U46619 in hPAs and rPAs, and by Ang II in rPAs, in an endothelium-dependent manner. AM251 did not influence vasoconstrictions induced by 5-HT or phenylephrine in rPAs. The monoacylglycerol lipase (2-arachidonoylglycerol degradation enzyme) inhibitor JZL184 (1 μM), but not the fatty acid amide hydrolase (anandamide degradation enzyme) inhibitor URB597 (1 μM) attenuated contractions evoked by U46619 in hPAs and rPAs, and Ang II in rPAs. 2-arachidonoylglycerol concentration-dependently induced relaxation of hPAs, which was inhibited by endothelium denudation or AM251 and enhanced by JZL184. Expression of CB₁ receptors was confirmed in hPAs and rPAs using Western blotting and immunohistochemistry. The present study shows the protective interaction between the endocannabinoid system and vasoconstriction to U46619 and Ang II in the human and rat pulmonary circulation. U46619 and Ang II may stimulate rapid endothelial release of endocannabinoids (mainly 2-arachidonoylglycerol), leading to CB1 receptor-dependent and/or -independent vasorelaxation, which in the negative feedback mechanism reduces later agonists-induced vasoconstriction.