Inhaled cysteinyl leukotrienes may cause recruitment of eosinophils into asthmatic airways. We compared the effects of inhaled leukotriene D4 (LTD4), methacholine, and allergen on airway eosinophils in 10 nonsmoking, atopic, mildly asthmatic subjects in a double-blind, diluent-controlled, randomized crossover study. Concentrations of LTD4, methacholine, and allergen resulting in a 30% decrease in FEV1, and diluent controls (ethanol and saline), were inhaled with at least 7 d between challenges. Spirometry was conducted for 4 h after inhalation challenge, and airway hyperresponsiveness (AHR) to methacholine was measured before and 24 h after challenge. Sputum was induced before and 4 h, 7 h, and 24 h after challenge. The maximum decrease in FEV1 was 31.4 ± 1.8% with LTD4, 39.4 ± 2.8% with methacholine, and 30.1 ± 3.4% with allergen. AHR to methacholine, at the provocative concentration causing a 20% decrease in FEV1 (PC20), was enhanced 24 h after allergen challenge, but remained unchanged 24 h after LTD4 and methacholine (p > 0.05). The percentage of eosinophils in sputum was increased after inhalation of allergen at 7 h and 24 h (p = 0.003), but not after LTD4 or methacholine (p = 0.70). We demonstrated that neither inhalation of LTD4 nor of methacholine at concentrations causing submaximal bronchoconstriction increases the number of sputum eosinophils in the airways of mildly asthmatic subjects. However, LTD4 may still be an important cofactor for eosinophil recruitment in asthma.