Background Nitric oxide (NO) is increased in the lung periphery of patients with chronic obstructive pulmonary disease (COPD). However the NO synthase(s) expression responsible for elevated NO has not been identified in the peripheral lung tissue of patients with COPD with varying severity. Methods Protein and mRNA expression of NO synthase type I (nNOS), type II (iNOS) and type III (eNOS) were quantified by Western blotting and RT-PCR, respectively, in specimens of surgically resected lung tissue from non-smoker controls, patients with COPD of varying severity, and smokers without COPD, or a lung epithelial cell line (A549). The effects of nitrative/oxidative stress on NOS expression and activity were also evaluated in vitro in A549 cells. nNOS nitration was quantified by immunoprecipitation and dimerization of nNOS was detected by low temperature SDS PAGE/Western blot in the presence of the peroxynitrite generator, SIN1, in vitro and in vivo. Results Lung tissue from patients with severe and very severe COPD had graded increases in nNOS (mRNA and protein) compared with non-smokers and normal smokers. Hydrogen peroxide (H₂O₂) and SIN1 as well as the cytokine mixture (IFN-, IL-1 and TNF-) increased mRNA expression and activity of nNOS in A549 cells in a concentration-dependent manner compared to non-treated cells. Tyrosine nitration resulted in an increase in nNOS activity in vitro, but did not affect its dimerization. Conclusions Patients with COPD have a significant increase in nNOS expression and activity that reflects the severity of the disease and may be secondary to oxidative stress.