The small GTP binding protein Rho and its downstream effector Rho-kinase are important regulators of vasoconstrictor tone. Rho-kinase is upregulated in experimental models of pulmonary hypertension and Rho-kinase inhibitors decrease pulmonary arterial pressure in rodents with monocrotaline and chronic hypoxia induced pulmonary hypertension. However less is known about responses when pulmonary vascular resistance is elevated on an acute basis by vasoconstrictor agents and hypoxia. In the present study iv injections of fasudil reversed pulmonary hypertensive responses to iv infusion of the thromboxane receptor agonist U-46619, ventilation with a 10% O₂ gas mixture and inhibited pulmonary vasoconstrictor responses to iv injections of angiotensin II, Bay K 8644 and U-46619 without prior exposure to agonists which can upregulate Rho-kinase activity. The calcium channel blocker isradipine and fasudil had similar effects and in small doses had additive actions in blunting vasoconstrictor responses suggesting parallel and series mechanisms. When pulmonary vascular resistance was increased with U-46619, fasudil produced similar decreases in pulmonary and systemic arterial pressure whereas isradipine produced greater decreases in systemic arterial pressure. The hypoxic pressor response was enhanced by L-NAME, 5-10 mg/kg iv, and fasudil or isradipine reversed the response to hypoxia in control and in L-NAME treated animals suggesting that the response is mediated by Rho-kinase and L-type Ca++ channels. These results suggest that Rho-kinase is constitutively active in regulating baseline tone and vasoconstrictor responses in the lung under physiologic conditions and that fasudil attenuates pulmonary vasoconstrictor responses to agents which act by different mechanisms without prior agonist exposure.