Rationale: The Acute Respiratory Distress Syndrome (ARDS)/Acute Lung Injury (ALI) was described 30 years ago, yet a definitive diagnosis for this syndrome remains difficult. The identification of appropriate biomarkers obtained from peripheral blood could provide additional non-invasive means for establishing a diagnosis in patients. Objective: To identify gene expression profiles that may be used to classify patients with ALI. Methods: 13 patients with ALI + sepsis and 20 patients with sepsis alone were recruited from the Medical Intensive Care Unit of the University of Pittsburgh Medical Center between February 2005 and June 2007. Whole blood was obtained from each patient within 48 hours of admission, and RNA was extracted for gene expression profiling. Several classification algorithms were used to develop a gene signature for ALI. This gene signature was subsequently validated in an independently obtained set of patients with ALI + sepsis (n = 8) and sepsis alone (n = 1). Measurements and Main Results: An eight-gene expression profile was found to be associated with ALI. Internal validation found that the gene signature was able to distinguish patients with ALI + sepsis from patients with sepsis alone with 100% accuracy, corresponding to a sensitivity of 100%, a specificity of 100%, a positive predictive value (PPV) of 100%, and a negative predictive value (NPV) of 100%. External validation of the gene signature was performed on an independently obtained set of patients. In the external validation set, the gene signature was able to distinguish patients with ALI + sepsis from patients with sepsis alone with 88.9% accuracy, corresponding to a sensitivity of 100%, a specificity of 50%, a PPV of 88.89%, and a NPV of 100%. Conclusions: The use of classification models to develop a gene signature from gene expression profiles provides a novel and accurate approach for classifying patients with ALI.