To study the rote of the inhibitory nonadrenergic noncholinergic (i-NANC) system in regulating bronchial reactivity during antigen challenge, we first tested a blocker of the i-NANC system (oxyhemoglobin, HbO2, 2.5μm) on the relaxation response of guinea pig tracheal strips (n=6) in vitro to electrical field stimulation (ES) in the presence of atropine (1μg/ml) and propranolol (2μg/ml). Fresh HbO2 significantly inhibited 35.3±4.5% (P<0.001) of the NANC relaxation response. Secondary, 26 anesthetized, ovalbumin-sensitized animals were divided into three groups: antigen challenged (n=10), pretreated with Hb02 (13 mg/kg) and challenged (n=9), and treated with HbO2 only (=7). Pulmonary resistance (RL) and dynamic compliance (Cdyn) were measured 15-20min prior to (baseline) and up to 30 min after antigen or HbO2 injection. Antigen challenge alone induced early maximal respiratory changes: RL increased 1646±115% above baseline (2 min) whereas Cdyn decreased 42±10% below baseline (4 min). These changes returned to baseline within 15 min. Pretreatment with Hb02 increased peak respiratory responses induced by antigen [RL, 3728±1680% above baseline; Cdyn, 69±7% below baseline (P<0.05). HbO2 delayed significantly (P<0.05) the time for recovery of RL and Cdyn. HbO2 alone had little effect on respiratory parameters. We conclude that HbO2 may antagonize the i-NANC system in the airway and this antagonism may accentuate pulmonary hypersensitivity during acute antigen challenge.