Pulmonary surfactant from bronchoalveolar lavages was obtained from 2 groups of horses. A control group consisting of 6 healthy racehorses that were paddock-rested and lavaged weekly for 6 consecutive weeks were compared with an experimental group of 10 healthy racehorses, lavaged weekly the same period, consisting of a 5 week incremental-intensity treadmill training programme and one week post training paddock rest. Phospholipid content of lavage fluid was determined indirectly by phosphorus assay, and surfactant functional activity was determined by bubble surfactometry. Total cell counts and differential cell percentages of lavage fluid were adjusted to reflect the dilution of alveolar epithelial lining fluid (ELF) using the lavage/serum urea ratio, and data were analysed per volume of ELF. There was no change in phospholipid content for either group, but some horses had consistently greater amounts than did others, ranging from 17.2–64.4 μg/μl. From the exercised group ELF had both increased nucleated cell numbers due to increased macrophage numbers, and increased numbers of erythrocytes. Surface tension increased significantly over the exercise protocol, but not in controls. Functional activity of surfactant varied between horses, independent of phospholipid content, with average values for individuals ranging 10.5–29.5 mN/m. We conclude that exercise of sufficient intensity to induce intrapulmonary haemorrhage also leads to functional decreases in surfactant activity, without affecting phospholipid content. This study also indicates that functional differences in surfactant exist between horses and may be a risk factor for development of exercise-induced pulmonary haemorrhage.