Endothelin-1 (ET-1) has recently been reported to have a potential pathophysiologic role in bronchial asthma. In the current study, we hypothesized whether ET-1 and a gene encoding ET-1 might be involved in airway hyperresponsiveness (AHR), which is a major feature of bronchial asthma. To test this hypothesis, we investigated airway responsiveness in ET-1^{+ / −} heterozygous knockout mice, which genetically produce lower levels of ET-1, and in ET-1^{+ / +} wild-type mice. Airway responsiveness was assessed through the concentration of an agonist required to double lung resistance (EC₂₀₀ Rl). Unexpectedly, airway responsiveness to methacholine was markedly enhanced in ET-1^{+ / −} heterozygous mice as compared with ET-1^{+ / +} wild-type mice (EC₂₀₀ Rl: 1.8 ± 0.1 versus 21.6 ± 5.6 mg/ml, p < 0.002). Pretreatment with the nitric oxide (NO) synthase inhibitor N^g-monomethyl-l-arginine (l-NMMA) significantly enhanced methacholine responsiveness in ET-1^{+ / +} wild-type mice, but not in ET-1^{+ / −} heterozygous mice. Meanwhile, there was no difference between ET-1^{+ / −} heterozygous mice and the wild-type mice in airway responsiveness to 5-hydroxytryptamine (5-HT). In sensitized mice, no significant differences in responsiveness to antigen were observed between the two groups. These findings suggest that the gene encoding ET-1 may be potentially involved in the etiology of airway hyperreactivity, and that the decrease in ET-1 concentration is associated with AHR to methacholine. In mice, ET-1 as well as NO may have a significant role in the homeostasis of airway physiology.