Methacholine inhalation tests were performed using a modification of the method described by Chai et al.12At the time of testing, all subjects had been free of acute respiratory tract infection or asthma exacerbation for a period of 4 weeks. Asthmatic children were asked to discontinue the use of inhaled β2-agonists (for 24 h), oral theophylline (for 48 h), and inhaled corticosteroids (for 7 days) before testing. Spirometric measurements (FEV1 and FVC) were made using a computerized spirometer (Microspiro-HI 298; Chest; Tokyo, Japan), in accordance with the recommendations of the American Thoracic Society (ATS).13 The time course of the preceding inspiration was standardized (ie, a rapid maximal inspiration without end-inspiratory pause), and the FVC maneuver was continued until a plateau in the volume-time curve display was obvious or exhalation exceeded 6 s. The subjects were trained for spirometry in a reproducible way (ie, a coefficient of variation of FEV1 < 5% in three consecutive flow-volume curves), and they were required to have a FEV1 of at least 80% of the predicted value.14 Methacholine (Sigma Diagnostics; St. Louis, MO) solution was prepared at different concentrations (0.075, 0.15, 0.3, 0.625, 1.25, 2.5, 5, 10, and 25 mg/mL) in buffered saline solution (pH 7.4). A Rosenthal-French dosimeter (Laboratory for Applied Immunology; Baltimore, MD), triggered by a solenoid valve set to remain open for 0.6 s, was used to generate an aerosol from a DeVilbiss 646 nebulizer (DeVilbiss Health Care; Somerset, PA), with air pressurized at 20 lb per square inch. Each subject inhaled five inspiratory capacity breaths of buffered saline solution and increasing concentrations of methacholine at 5-min intervals. This gave an output of 0.009 ± 0.0014 mL (mean ± SD) per inhalation. FEV1 and FVC were measured 90 s after inhalation at each concentration level, and the largest value of triplicate FEV1 or FVC was used for the analysis. The procedure was terminated when the FEV1 decreased by > 20% of its post-saline solution value or when the highest methacholine concentration (25 mg/mL) was reached. The percentage fall in FEV1 from the post-saline solution value was plotted against the log concentration of the inhaled methacholine. PC20 was calculated by interpolating between two adjacent data points. The ΔFVC relative to baseline FVC after saline solution inhalation was also calculated using a log-linear interpolation.