Rationale and Objectives. To investigate changes in lung microstructure in healthy adult subjects with no smoking history using diffusion-weighted 3He MRI. Materials and Methods. Diffusion magnetic resonance imaging using hyperpolarized helium 3 ( 3He) was applied to healthy volunteers to explore the dependence of lung microstructural changes with age, reflected by changes in the apparent diffusion coefficient (ADC) of 3He in lung air spaces. Data from three sites (University of Virginia (UVa), N = 25; University of Wisconsin (UW), N = 8; University of Nottingham (UN), N = 11) were combined in pooled analysis, including a total of N = 44 subjects (age range, 18-69 years; average age, 41.7 ± 16.7 years). Results. ADC was found to depend on age at all three sites (UW, R = +0.95, P = .0003; UVa, R = +0.74, P < .0001; UN, R = +0.96, P < .0001). Increases in mean ADCs with age appeared similar across sites (UW, +0.0017 cm 2s -1y -1; UVa, +0.0015 cm 2s -1y -1; pooled, +0.0015 cm 2s -1y -1; P = .71). In a regional analysis performed on UW data, the increase in ADC affected all regions of the lung, but the apical and middle regions showed a greater increase compared with the base of the lung. Conclusion. Results suggest the observed age dependence of the ADC may be caused by changes in lung microstructure that increase alveolar volume during the aging process.