We have previously found an age-dependent relaxing effect of furosemide in normal fetal, newborn, and adult guinea pig airways with fetal trachea exhibiting the greatest relaxation and adult tissue the least. This study was designed to expand upon this finding by determining ifin vivohyperoxic exposure would influencein vitroairway relaxation mediated by the loop diuretics, furosemide and ethacrynic acid, and the β2-adrenoceptor agonist, salbutamol. Newborn guinea pigs were raised in >95% Fi02until ill; controls in room air. Isometric relaxation to 3 × 10−5M furosemide, 3 × 10−6M ethacrynic acid, or 10−8−10−6M salbutamol was recorded in 3 × 10−6M histamine-constricted airway rings. Ethacrynic acid, like furosemide, relaxed newborn guinea pig airways. Hyperoxia did not alter the contractile effect of 3 × 10−6M histamine but did significantly decrease the relaxing effect of furosemide, ethacrynic acid, and salbutamol. Loop diuretic mediated airway relaxation was accentuated in HEPES buffer when compared with Krebs, whereas salbutamol-mediated relaxation was unaffected. These results suggest that hyperoxia nonspecifically decreases airway responsiveness to the relaxing agents studied.