The aim of the present study was to determine: 1) whether 40-50 ppm nitric oxide (NO) increases diffusing capacity of the lung for NO (DL,NO) and carbon monoxide (DL,CO), membrane diffusing capacity for CO (Dm,CO) and pulmonary capillary blood volume (Vc); 2) the actual number of tests required to provide a reasonable estimate of DL,NO, DL,CO, Dm,CO and Vc; and 3) repeatability of these parameters using the single-breath DL,NO-DL,CO method.In total, 31 subjects performed five single-breath hold manoeuvres at rest, inhaling 43¡3 ppm NO together with a standard diffusion mixture.DL,NO (Dm,CO) remained unchanged from the first to fifth trial. However, compared with the first trial, DL,CO and Vc had decreased by the fourth (-4¡5%; 95% confidence interval (CI)5-5--2%) and third trial (-5¡7%; 95% CI5-7--2%), respectively. Repeatability over five trials was 17, 3 and 7 mL?min -1?mmHg -1 for DL,NO, DL,CO and Dm,CO, respectively, and 13 mL for Vc when Dm,CO5DL,NO/2.42.In conclusion, nitric oxide inhaled during sequential single-breath manoeuvres has no effect on diffusing capacity of the lung for nitric oxide and, thus, membrane diffusing capacity for carbon monoxide. Since more than two and three trials will lower pulmonary capillary blood volume and diffusing capacity of the lung for carbon monoxide, respectively, the average value of only two properly performed trials is suggested.