Nitric oxide (NO) may serve as a lipophilic chain braking antioxidant (J Biol Chem 269:26066, 1994). On the other hand, NO may have an adverse effect on the surfactant system, as it may form peroxynitrite that causes lipid peroxidation and nitration of surfactant proteins (Am J Physiol 265:L555, 1993). Cyclic exposure of surfactant to the air-liquid interface, as occurs during breathing, leads to degradation of surfactant from dense, protein-rich large aggregates to less dense, protein poor small vesicles that no longer are surface active (Am J Physiol 262:L242, 1992). The aim was to investigate whether NO affects the aggregate conversion rate of lung surfactant. The air-liquid area of a buffered Ca++-saline mixture, containing surfactant (0.5 mM phospholipid), was cycled between 0.7 and 1.4 cm2 at a frequency of 0.4 Hz for 4 to 12 hrs. NO (80 ppm), introduced into the vessel in 20% O2, decreased the conversion rate of the dense surfactant aggregates to less dense vesicles by 65±4%, as compared with 20% O2. The surface cycling was required for the expression of the activity of NO. The protective effect of NO was also seen in surfactant that did not contain SP-A. The aggregate conversion during surface cycling and the inhibitory effect of NO on the aggregate conversion was evident in the absence of protease activity. However, NO had no effect when synthetic phospholipids were cycled, or when the cycling took place in the absence of oxygen. During surface cycling in air, addition of NO decreased the formation of malondialdehyde by 75±9%. We propose that NO has a role in regulating the rate of conversion of the large surfactant aggregates to smaller vesicles that are not surface active. This effect may be related to the role of NO as a lipophilic antioxidant preventing oxidative inactivation of interfacial surfactant film. The biological significance of the effect of NO in decreasing the rate of the aggregate conversion during surface cycling, remains to be studied.