<p>TGF-β1-induced lung injury initially results in surfactant dysfunction and alveolar instability (microatelectases). Morphology showed that at end-expiratory pressure of 2cmH₂O the burden of microatelectasis was high but could be mitigated with a pressure of 8cmH₂O by 35% without undue strain. Hence, we hypothesized that ventilation of lungs with a higher burden of microatelectasis at positive end-expiratory pressure (PEEP) of 2cmH₂O triggers more pronounced injury progression compared to PEEP=8cmH₂O. Mice were randomized to receive either TGF-β1 (AdTGF-β1) for injury induction or empty control vector (AdCl). After a second randomization one week later, mice were ventilated for 4&#xa0;h with PEEP = 2 or 8cmH₂O resulting in 4 experimental groups: AdTGF-β1-PEEP2 (<i>n</i> = 11), AdTGF-β1-PEEP8 (<i>n</i> = 10), AdCl-PEEP2 (<i>n</i> = 11) and AdCl-PEEP8 (<i>n</i> = 10). During ventilation, every 30&#xa0;min, Quick-Prime (tissue elastance) and Snap-Shot (dynamic compliance) measurements were performed immediately before and after deep inflations. Finally, lungs were either fixed for stereology (both PEEP2 groups <i>n</i> = 6/ both PEEP 8 groups <i>n</i> = 5) or broncho-alveolar lavage (<i>n</i> = 5) and tissue harvest for transcriptome analyses. During ventilation the increase in tissue elastance was largest in AdTGF-β1-PEEP2 group but reversible by deep inflations. Finally, both AdTGF-β1-groups showed comparable worsening in lung mechanics, and BAL-albumin and neutrophils were elevated. Quantitative morphology showed no differences and transcriptome minimal differences attributable to PEEP-level. Low-PEEP ventilation of AdTGF-β1 lungs with a high burden of microatelectases induced lung mechanical worsening most likely due to progressive alveolar derecruitment. This injury pattern was reversible upon deep inflations and not linked with signs of persistent lung injury such as vascular leakage, edema or inflammation.</p>