Rationale: Ventilator-induced lung injury has been predominantly studied in adults. Objectives: To explore the effects of age and lung development on susceptibility to such injury. Methods: Ex vivo isolated nonperfused rat lungs (infant, juvenile, and adult) were mechanically ventilated where VT was based on milliliters per kilogram of body weight or as a percentage of the measured total lung capacity (TLC). In vivo anesthetized rats (infant, adult) were mechanically ventilated with pressure-limited VTs. Allocation to ventilation strategy was randomized. Measurements: Ex vivo injury was assessed by pressure–volume analysis, reduction in TLC, and histology, and in vivo injury by lung compliance, cytokine production, and wet- to dry-weight ratio. Main Results: Ex vivo ventilation (VT 30 ml · kg⁻¹) resulted in a significant reduction (36.0 ± 10.1%, p < 0.05) in TLC in adult but not in infant lungs. Ex vivo ventilation (VT 50% TLC) resulted in a significant reduction in TLC in both adult (27.8 ± 2.8%) and infant (10.6 ± 7.0%) lungs, but more so in the adult lungs (p < 0.05); these changes were paralleled by histology and pressure–volume characteristics. After high stretch in vivo ventilation, adult but not infant rats developed lung injury (total lung compliance, wet/dry ratio, tumor necrosis factor α). Surface video microscopy demonstrated greater heterogeneity of alveolar distension in ex vivo adult versus infant lungs. Conclusion: These data provide ex vivo and in vivo evidence that comparable ventilator settings are significantly more injurious in the adult than infant rat lung, probably reflecting differences in intrinsic susceptibility or inflation pattern.