Nebulized amiloride (AMIL) is used in cystic fibrosis (CF) to block Na+ hyperabsorption and dehydration of mucus. CF patients often have inflammation and reactive airways. Bovine and canine airways relax to AMIL in vitro, suggesting a benefit as a bronchodilator, but we found (Pediatr Res 1996;39:387A) that guinea pig (GP), mouse, and human fetal airways contract to AMIL when tone is induced by acetylcholine (ACh, neurotransmitter), independent of airway epithelium. However, the effect of AMIL on airway tone induced by inflammatory mediators, such as leukotriene D4 (LTD4), is not known. Thus, we further defined the mechanism of AMIL-mediated airway contraction, and compared this to the response on airway tone induced by other agonists. Methods. GP (n=6-16), mouse (n=5-9), and human fetal (6 fetuses, 11-16 wks gestation) airway ring segments were constricted with an EC50-75 of ACh, KCl, or LTD4. Changes in isometric tension to cumulative additions of AMIL (0.01-100 μM) were measured. Results. Developmental regulation was not detected. AMIL-mediated contraction was attenuated by inhibition of protein kinase C (PKC) with GF109203X (p<0.01) and staurosporine (p<0.01). However, after constriction with KCl, AMIL relaxed GP (108±15%), mouse (57±32%), and human fetal airway tone (137±58%), and after constriction with LTD4, AMIL relaxed GP(82±10%) and human fetal airway tone (62±23%). Mice did not respond to LTD4. Conclusions. After constriction with ACh, airways from all three species contracted to AMIL in vitro, and this contraction may be mediated by PKC. However, after constriction with KCl and LTD4, AMIL caused relaxation. AMIL may be useful clinically as a bronchodilator when airway hyperresponsiveness is mediated by leukotrienes. Funded by: US Army HSC, NIH RR/AI11091, and NIH HL-45220.