Although the pathogenic and genetic basis of acute lung injury (ALI) remains incompletely understood, the identification of novel ALI biomarkers holds promise for unique insights. Expression profiling in animal models of ALI (canine and murine) and human ALI detected significant expression of pre-B-cell colony enhancing factor (PBEF), a gene not previously associated with lung pathophysiology. These results were validated by real-time polymerase chain reaction and immunohistochemistry studies with PBEF protein levels significantly increased in both bronchoalveolar lavage fluid and serum of ALI models as well as in cytokine- or cyclic stretch-activated lung microvascular endothelium. We genotyped 2 single nucleotide polymorphisms (SNPs) in a well-characterized Caucasian sample of sepsis-associated ALI, severe sepsis, and healthy subjects and observed that carriers of the haplotype GC from SNPs T-1001G and C-1543T had a 7.7-fold higher risk of ALI (95% CI, 3.01 -19.75, p < 0.001). The T variant from the SNP C-1543T resulted in a significant decrease in the transcription rate (1.8 fold, p <0.01) by the reporter gene assay. Together, these results strongly indicate that PBEF is a potential novel biomarker in ALI and demonstrate the successful application of robust genomic technologies in the identification of candidate genes in complex lung disease.