Mechanical ventilation is a life-saving intervention in patients with respiratory failure. However, human and animal studies have demonstrated that mechanical ventilation using large tidal volumes ( 12 ml/kg) induces a potent inflammatory response and can cause acute lung injury. We hypothesized that mechanical ventilation with a "non-injurious" tidal volume of 10 ml/kg would still activate a transcriptional program that places the lung at risk for severe injury. To identify key regulators of this transcriptional response, we integrated gene expression data obtained from whole lungs of spontaneously breathing mice and mechanically ventilated mice with computational network analysis. Topological analysis of the gene product interaction network identified Jun and Fos family of proteins as potential regulatory hubs. Electrophoretic mobility gel shift assay confirmed protein binding to AP-1 consensus sequences, and supershift experiments identified JunD and FosB as components of ventilation-induced AP-1 binding. Specific recruitment of JunD to the regulatory region of the F3 gene by mechanical ventilation was confirmed by chromatin immunoprecipitation assay. In conclusion, we demonstrate a novel computational framework to systematically dissect transcriptional programs activated by mechanical ventilation in the lung, and show that non-injurious mechanical ventilation initiates a response that can prime the lung for injury from a subsequent insult.