The hepatopulmonary syndrome (HPS) results from intrapulmonary vasodilation in the setting of cirrhosis and portal hypertension. In experimental HPS, pulmonary endothelial endothelin B (ETB) receptor overexpression and increased circulating endothelin-1 (ET-1) contribute to vasodilation through enhanced endothelial nitric oxide synthase (eNOS)-derived nitric oxide (NO) production. In both experimental cirrhosis and prehepatic portal hypertension, ETB receptor overexpression correlates with increased vascular shear stress, a known modulator of ETB receptor expression. We investigated the mechanisms of pulmonary endothelial ETB receptor-mediated eNOS activation by ET-1 in vitro and in vivo. The effect of shear stress on ET B receptor expression was assessed in rat pulmonary microvascular endothelial cells (RPMVECs). The consequences of ETB receptor overexpression on ET-1-dependent ETB receptor-mediated eNOS activation were evaluated in RPMVECs and in prehepatic portal hypertensive animals exposed to exogenous ET-1. Laminar shear stress increased ETB receptor expression in RPMVECs without altering mRNA stability. Both shear-mediated and targeted overexpression of the ETB receptor enhanced ET-1-mediated ETB receptor-dependent eNOS activation in RPMVECs through Ca2+-mediated signaling pathways and independent of Akt activation. In prehepatic portal hypertensive animals relative to control, ET-1 administration also activated eNOS independent of Akt activation and triggered HPS. These findings support that increased pulmonary microvascular endothelial ETB receptor expression modulates ET-1-mediated eNOS activation, independent of Akt, and contributes to the development of HPS.