Leukotriene (LT) D4 is a potent constrictor of human airways and a putative mediator of asthma. However, its mechanism of action in man has not been established. In the present study we sought to determine the role of upper airway reflexes, cholinergic pathways, and cyclooxygenase products of arachidonic acid metabolism in mediating LTD4-induced bronchoconstriction in man. Six normal subjects underwent bronchoprovocation testing with LTD4 after pretreatment with either aerosolized phosphate-buffered saline, aerosolized atropine (1.5 mg), aerosolized lidocaine (80 to 160 mg), or oral indomethacin (50 mg three times daily for 10 doses). Specific airway conductance (SGaw), the flow rate at 30% of vital capacity from a partial forced expiratory maneuver (V30P), and the FEV1 were measured at each concentration of LTD4. We calculated the provocative concentration of LTD4 required to produce a 35% fall in SGaw (PC35SGaw) or a 30% fall in V30P (PC30V30P) and the slope of the LTD4 dose-response curve. Atropine increased baseline SGaw 49% (p less than 0.01) and V30P 43% (p less than 0.005). Atropine also increased the PC35SGaw and PC30V30P and increased the slope of the LTD4 dose-response curve. However, neither of these opposing effects was significant. Lidocaine had no effect on either baseline function or the airway response to LTD4. Indomethacin produced small decreases in baseline V30P (12%) and FEV1 (3%) (p less than 0.05 for both), but it too had no effect on the airway response to LTD4. These results indicate that the bronchoconstriction produced by aerosolized LTD4 in normal subjects is not mediated by cyclooxygenase products of arachidonic acid metabolism or irritant receptors in the upper airways. Although cholinergic pathways may play some role, the data suggest that bronchoconstriction results, at least in part, from a direct effect of LTD4 on airway smooth muscle.