Low viscosity of the surface of alveolar fluid is mandatory for undisturbed surfactant function. Based on the known reduction of the viscosity of surfactant-like phospholipid (PL-) mixtures by plasmalogens, the effect of cholesterol and surfactant protein (SP-) B on surface viscosity of these lipid mixtures has been studied. Surface viscosity at the corresponding surface tension was measured with the oscillating drop surfactometer. We found that the viscosity was lowest in cholesterol-, followed by plasmalogen- and SP-B containing samples. Addition of SP-B to a plasmalogen-containing PL-mixture caused a further decrease in viscosity. However, in cholesterol containing mixtures, addition of SP-B led to a significant increase in viscosity, and the effect was reversed by further addition of plasmalogens. We conclude that SP-B, plasmalogens and cholesterol all affect the surface viscosity, thus synergistically regulate monolayer stability. This suggests that they are all needed in vivo for fine tuning of surface properties of pulmonary surfactant.