Nitric oxide (NO) is a major regulatory molecule of the cardiovascular system; however, measurement of vascular NO synthesis in vivo represents a major challenge. NO stemming from the lower respiratory tract has been used as a marker of vascular endothelial function. Experimental evidence for this concept is lacking. Therefore, the aim of the present study was to investigate this relationship. Lower respiratory tract exhaled NO concentration, together with systemic and pulmonary artery pressure, was measured in endothelial nitric oxide synthase (NOS) (eNOS) null mice (eNOS-/-). Similar studies were performed in inducible NOS (iNOS) null mice (iNOS-/-). Defective endothelial NO synthesis in eNOS-/- mice (evidenced by systemic and pulmonary hypertension) was associated with augmented exhaled NO levels (12.5 +/- 1.9 versus 9.8 +/- 1.2 parts per billion (ppb), eNOS-/- versus wild type), whereas normal endothelial NO synthesis in iNOS-/- mice was associated with decreased exhaled NO levels (4.3 +/- 1.5 ppb). Augmented exhaled NO levels in eNOS-/- mice were associated with upregulation of iNOS expression in the lung. These results indicate that inducible nitric oxide synthase is a major determinant of gaseous nitric oxide production in the lung, and lower respiratory tract exhaled nitric oxide does not always represent a marker of vascular endothelial nitric oxide synthesis.