Objective: To assess repeatability and reproducibility of spirometry measurements, and bronchodilator responsiveness (BDR), in healthy 3-6-year-old preschool children and those with asthma. Design: Spirometry was performed before and 20 minutes after administering either inhaled placebo (for repeatability) or 400 μg salbutamol (for BDR) on two separate occasions (reproducibility) 3-23 days apart in asthmatic preschoolers and healthy controls. Settings: Lung Function Laboratory, Hospital de Dona Estefania, Lisbon. Participants: Healthy preschool children and those with physician-diagnosed asthma, recruited from local Health Clinics and Outpatient Clinic. Main outcome measures: Paired measurements of forced expired volume in 0.75 s (FEV0.75) and forced mid-expiratory flows (FEF 25-75). Results: Technically successful baseline results were obtained in 86% of children assessed. Paired data were obtained in 43 asthmatic and 22 controls (median (range) age: 5.1 (3.4-6.8) years). Baseline FEV 0.75 was significantly lower in asthmatic children (mean (SD): 90 (15)% predicted) than in controls (102 (13) % predicted; p<0.001). Within-occasion coefficient of repeatability following placebo was similar in both groups, being 10.4% in asthma and 13.2% in controls for FEV0.75. Following bronchodilator, FEV0.75 increased significantly more in asthmatic preschoolers (mean (SD): 15.0 (12) %) than in controls (4.5 (5) %; p<0.001), with no significant difference between groups post-bronchodilator. Between-occasion variability was similar to within-day repeatability in controls, but almost twice as high in asthmatic children. Conclusions: BDR can be assessed reliably using FEV0.75 in wheezy preschoolers, provided within-subject variability and responsiveness in health are taken into consideration.