We investigated acute effects of inhalation of hypertonic saline solution (HSS) and oxygen (O-2, control exposure) on pulmonary diffusing capacity for nitric oxide (DLNO) and carbon monoxide (DLCO). In a randomized crossover study, 20 healthy, non-smoking subjects were allocated to short-term inhalation of HSS or O-2. Spirometry [(forced expiratory volume in 1 s (FEV1) and forced vital capacity (FVC)] and combined single breath DLNO-DLCO measurements were performed before and immediately after inhalation of either HSS or O-2. Percent changes were presented as median values (interquartile range). After HSS inhalation, DLNO, FEV1 and FVC were decreased by -3.0% (-7.3, 0.5), -3.1% (-4.2, -1.6) and -1.2% (-3.3, 0.6), respectively (all P < 0.05), without significant effect on DLCO. No changes in spirometry and diffusing capacity were observed following O-2 inhalation. Acute inhalation of HSS causes a slight decrease in membrane conductance, probably as a result of fluid imbalance at the alveolar surface and interstitial fluid accumulation, both of which could impair gas exchange.