Pulmonary diffusing capacity of NO (Dl NO) was determined by performing single-breath experiments on six anesthetized paralyzed supine rabbits, applying inspiratory concentrations of NO (Fi NO) within a range of 10 parts per million (ppm) ≤ Fi NO ≤ 800 ppm. Starting from residual volume, the rabbit lungs were inflated by 50 ml of a NO-nitrogen-containing indicator gas mixture. Breath-holding time was set at 0.1, 1, 3, 5, and 7 s. Alveolar partial pressure of NO was determined by analyzing the end-tidal portion from expirates, with the use of respiratory mass spectrometry. In the six animals, pulmonary diffusing capacity of NO averaged Dl NO = 1.92 ± 0.21 ml ⋅ mmHg−1 ⋅ min−1(mean ± SD value). Despite extreme variations in Fi NO, we found very similar Dl NOvalues, and in three rabbits we found identical values even at such different Fi NO levels of 80 ppm or 500, 20, or 200 ppm as well as 10 or 800 ppm. There was also no dependence of Dl NO on the respective duration of the single-breath maneuvers. In addition, the time course of NO removal from alveolar space was independent of applied Fi NOlevels. These results suggest that Dl NOdeterminations are neither affected by chemical reactions of NO in alveolar gas phase as well as in lung tissue nor biased by endogenous release of NO from pulmonary tissue. It is our conclusion that the single-breath diffusing capacity of NO is able to provide a measure of alveolar-capillary gas conductance that is not influenced by the biochemical reactions of NO.