Abstract Background: Recent studies in healthy mice and rats have reported that positive pressure ventilation delivered with physiological tidal volumes at normal end-expiratory volume worsens lung mechanics and induces cytokine release, thus suggesting that detrimental effects are due to positive pressure ventilation per se. The aim of this study in healthy animals is to assess whether these adverse outcomes depend on the mode of mechanical ventilation. Methods: Rats were subjected to 4 h of spontaneous, positive pressure, and whole-body or thorax-only negative pressure ventilation (N = 8 per group). In all instances the ventilatory pattern was that of spontaneous breathing. Lung mechanics, cytokines concentration in serum and broncho–alveolar lavage fluid, lung wet-to-dry ratio, and histology were assessed. Values from eight animals euthanized shortly after anesthesia served as control. Results: No evidence of mechanical ventilation–dependent lung injury was found in terms of lung mechanics, histology, or wet-to-dry ratio. Relative to control, cytokine levels and recruitment of polymorphonuclear leucocytes increased slightly, and to the same extent with spontaneous, positive pressure, and whole-body negative pressure ventilation. Thorax-only negative pressure ventilation caused marked chest and lung distortion, reversible increase of lung elastance, and higher polymorphonuclear leucocyte count and cytokine levels. Conclusion: Both positive and negative pressure ventilation performed with tidal volumes and timing of spontaneous, quiet breathing neither elicit an inflammatory response nor cause morpho-functional alterations in normal animals, thus supporting the notion of the presence of a critical volume threshold above which acute lung injury ensues. Distortion of lung parenchyma can induce an inflammatory response, even in the absence of volotrauma.