The monolayer properties of two different sphingomyelin samples from bovine milk (one pure with respect to sphingomyelin (>99%) and one sphingolipid fraction containing 68% sphingomyelin) at the air/water interface and the interaction between the lipid monolayer and xanthine oxidase (XO) from the milk fat globule membrane were investigated by the film balance technique. For comparison, similar measurements were performed on distearoylphosphatidylcholine (DSPC) monolayers. Bovine milk sphingomyelin formed monolayers comparable with those of other natural sphingomyelins. A liquid-condensed phase transition at 20-22 mN/m reflected the high amount of long saturated fatty acids, as in the case for monolayers of egg sphingomyelin. Monolayers of bovine milk sphingomyelin were metastable, as opposed to those of DSPC. The observed decrease in surface area with time at constant pressure indicated that dissolution/expulsion into the subphase took place. A pure sphingomyelin monolayer was significantly more stable at a surface pressure of 10 mN/m than at 20 mN/m. The discontinuity in the surface area change versus time and the low solubility of the lipid show that the instability cannot be explained by simple desorption of lipid monomers. The presence of XO in the subphase increased the maximal surface pressure at an area per molecule of sphingomyelin of 30 A2 (maximal compression) by 15 mN/m for the sphingolipid sample and 20 mN/m for pure sphingomyelin, indicating a stabilization of sphingomyelin monolayers in the presence of XO at high surface pressure. This effect was not observed for DSPC monolayers, which suggests a specific interaction between sphingomyelin and XO.