To gain insight into the source of exhaled nitric oxide (NO) in normal humans, we examined the effects of respiratory pattern on the concentration of NO in exhaled air while subjects were wearing a noseclip and then under endotracheal intubation, using a specially designed gas sampling system to separate exhaled air into two fractions: the initially exhaled 200 ml (Fraction 1; F1), and the remainder (Fraction 2; F2). We also examined the effect of hypoxic gas inhalation (F(I)O2 = 0.1, 3 min) on the concentration of exhaled NO. The concentration of exhaled NO measured with a chemiluminescence NO analyzer was significantly lower with intubation, by 59.2 +/- 10.6% (mean +/- SD) (F1) and 54.4 +/- 8.0% (F2), than without intubation. The concentration of NO in F1 was consistently higher than that in F2 with or without intubation. With breath holding, the concentration of NO increased markedly only in F1. In contrast, prolongation of the expiratory phase slightly but significantly increased the concentration of NO only in F2. Inhalation of hypoxic gas did not cause any significant change in NO concentration in either fraction. These data indicate that in normal humans wearing a noseclip, about 40 to 45% of NO in exhaled air originates from the lungs, particularly from intrathoracic airways. The concentration of exhaled NO collected from subjects wearing a noseclip is not affected by hypoxic gas inhalation.