Background Pulmonary function tests are central to diagnosis and monitoring of respiratory diseases but do not provide information on regional lung function heterogeneity. Fluorine 19 (19F) MRI of inhaled perfluoropropane permits quantitative and spatially localized assessment of pulmonary ventilation properties without tracer gas hyperpolarization. Purpose To assess regional lung ventilation properties using 19F MRI of inhaled perfluoropropane in participants with asthma, participants with chronic obstructive pulmonary disease (COPD), and healthy participants, including quantitative evaluation of bronchodilator response in participants with respiratory disease. Materials and Methods This prospective, dual-center study included participants with asthma or COPD from July 2019 to September 2022 and healthy participants from May 2018 to June 2019. Participants underwent conventional spirometry, proton MRI, and 19F MRI following inhalation of a 79% perfluoropropane and 21% oxygen gas mixture. Three-dimensional 19F MRI scans were acquired during a single breath hold. For participants with asthma or COPD, spirometric and MRI measurements were repeated following administration of nebulized salbutamol. Ventilation defect percentage (VDP) was calculated from perfluoropropane distribution. Linear mixed-effects models were used to assess differences in VDP between participant groups and before and after bronchodilator administration. Results Thirty-five participants with asthma (mean age, 50 years ± 18 [SD]; 21 male participants), 21 participants with COPD (mean age, 69 years ± 6; 14 male participants), and 38 healthy participants (mean age, 41 years ± 11; 20 male participants) were evaluated. 19F MRI-derived VDP was elevated in participants with COPD (geometric mean, 27.2%) and participants with asthma (geometric mean, 8.3%) compared with healthy participants (geometric mean, 1.8%; geometric mean ratio, 15.2 [95% CI: 11.1, 20.6] for COPD and 4.6 [95% CI: 3.2, 6.6] for asthma; P < .001 for both). After bronchodilator administration, VDP was reduced by 33% in participants with asthma (from 8.3% to 5.6%) and 14% in participants with COPD (from 27.2% to 23.3%; P < .001 for both). Conclusion 19F MRI of inhaled perfluoropropane was sensitive to changes in regional ventilation properties associated with lung disease and enabled quantification of changes following bronchodilator therapy. Published under a CC BY-NC-ND 4.0 license. Supplemental material is available for this article. See also the editorial by Unger in this issue.