The predominant airway site and mechanism underlying ozone (O3)-induced respiratory hyperresponsiveness was examined in anesthetized guinea pigs and in vitro tissue preparations. Animals exposed to 1.0 or 1.2 ppm O3 (1 h) demonstrated an enhanced airway response to subcutaneous histamine compared with air-exposed animals. The anatomic site of hyperresponsiveness most likely did not involve the parenchyma, since quasi-static deflationary pulmonary compliance was decreased to a similar extent by histamine in air- and O3-preexposed animals. In contrast, the conducting airways were probably involved as changes in pulmonary resistance elicited by subcutaneous histamine were greater in O3- than in air-exposed animals. Neither atropine nor vagotomy abolished this enhanced responsiveness induced by O3. Although vagal interruption did not alter responsiveness, O3-exposed animals demonstrated greater respiratory responses to efferent electrical stimulation of the vagi than air-exposed animals. This suggests the site of hyperresponsiveness may be located distal to the site of efferent stimulation, possibly in the smooth muscle itself or in its microenvironment.