Endothelin-1 (ET-1) is a 21-amino acid peptide synthesized by several cell types in the lung. Locally, ET-1 regulates vascular and airway tone and is mitogenic for vascular and airway smooth muscle cells. Little, however, is known about the regulation of ET-1 in pulmonary endothelial cells. Cultured rat lung endothelial cells (RLECs) release significant amounts of ET-1 into the supernatant, and isolation of RNA followed by reverse transcription and polymerase chain reaction amplification confirms the presence of ET-1 mRNA. Exposure of RLECs to a hypoxic environment for 24 h decreases ET-1 production by approximately 50% compared with normoxic controls. The effect of hypoxia is reversible upon restoration of a normoxic environment. RNase protection studies reveal decreased ET-1 mRNA in hypoxic cells. Inhibition of nitric oxide (NO) synthase increases ET-1 synthesis during normoxia and hypoxia without altering the inhibitory effect of hypoxia. The addition of 10% carbon monoxide (CO) to the hypoxic environment does not erase the effect of hypoxia on ET-1 production, suggesting that the transduction process does not involve a heme sensor. In summary, we conclude that 1) RLECs synthesize ET-1; 2) hypoxia reversibly decreases ET-1 production; 3) constitutive NO production decreases ET-1 release during normoxia and hypoxia; 4) inhibiting constitutive NO synthesis does not prevent the decrease in ET-1 release caused by hypoxia; and 5) this effect of hypoxia appears to be transduced without the involvement of a heme sensor.