OBJECTIVE To determine cardiorespiratory responses of Thoroughbreds to uphill and downhill locomotion on a treadmill at identical gradients. ANIMALS 5 highly trained Thoroughbred geldings. PROCEDURES Thoroughbreds were exercised for 2-minute intervals on a treadmill at 1.7, 3.5, 6.0, 8.0, and 10.0 m/s at a 4% incline, 0% incline (horizontal plane), and 4% decline in random order on different days. Stride frequency, stride length, and cardiopulmonary and O2-transport variables were measured and analyzed by means of repeated-measures ANOVA and Holm-Šidák pairwise comparisons. RESULTS Horses completed all treadmill exercises with identical stride frequency and stride length. At identical uphill speeds, they had higher (vs horizontal) mass-specific O2 consumption (mean increase, 49%) and CO2 production (mean increase, 47%), cardiac output (mean increase, 21%), heart rate (mean increase, 11%), and Paco2 (mean increase, 1.7 mm Hg), and lower Pao2 (mean decrease, 5.8 mm Hg) and arterial O2 saturation (mean decrease, 1.0%); tidal volume was not higher. Downhill locomotion (vs horizontal) reduced mass-specific O2 consumption (mean decrease, 24%), CO2 production (mean decrease, 23%), and cardiac output (mean decrease, 9%). Absolute energy cost during uphill locomotion increased linearly with speed at approximately twice the rate at which it decreased during downhill locomotion. CONCLUSIONS AND CLINICAL RELEVANCE Findings suggested that for Thoroughbreds, downhill locomotion resulted in a lower energy cost than did horizontal or uphill locomotion and that this cost changed with speed. Whether eccentric training induces skeletal muscle changes in horses similar to those in humans remains to be determined.