Neutrophil elastase (NE) is a powerful serine protease capable of degrading most protein components of the extracellular matrix. We hypothesize that this elastase may play a significant role in lung cancer development and tested our hypothesis in a study of 348 primary lung cancer cases and 299 controls. Analysis of the entire gene using denaturing high performance liquid chromatography identified two novel single nucleotide polymorphisms (SNPs) in the promoter region: -903 T or G (REP-a) and -741 G or A (REP-b). Allele frequencies of these two SNPs were compared between the cases and controls using chi(2) statistics. The estimated relative risk in association with the TT at REP-a or the GG at REP-b was measured by odds ratio. Individuals with -903TT or -741GG allele had a 2.3 and 1.4 times higher risk of developing lung cancer than those with TG or AA+AG alleles, respectively. The relative risk for the combined effects of both high-risk alleles at REP-a and REP-b, i.e., TT-GG type, was 24.8. Functional association of the two markers with cancer risk was examined by luciferase activity of the promoter containing different SNPs. We demonstrated a 1.9-fold relative luciferase activity in the promoter construct with -903T/-741G (T-G) compared with the -903G/-741A (G-A) in A549 human non-small cell lung cancer cells, providing evidence that the TT-GG type correlates with a high NE level. In conclusion, our findings support an etiological role of NE in lung cancer development.