Four isocaloric diets containing 4, 8, 12 and 16% dietary fat (as soybean oil) were fed to four horses at four intervals according to a Latin square design. After 3 weeks of conditioning at each interval, diet effects were evaluated by trotting all horses at 3.2 m/sec for 6 hours. Pre- and posttrotting responses were measured in muscle and liver glycogen, serum long-chain fatty acids, serum electrolytes, serum enzymes, serum cholesterol, plasma glucose, packed cell volume and hemoglobin. Dietary fat was highly correlated with exercise-induced plasma glucose changes and with cholesterol concentrations. Regardless of the diet, linoleate concentration was about eight times higher than that of the other fatty acids, and it increased slightly as dietary fat levels increased. Stearate concentration also increased with increasing dietary fat but palmitic and oleic acid decreased. Increases in fat intake also resulted in slight increases in liver glycogen at the resting level. Conditioning resulted in a significant decrease in exercise-induced fluctuations of serum enzymes and electrolytes but significantly increased elevations of plasma long-chain fatty acid concentrations. Resting muscle glycogen increased by 37% during the study as a result of conditioning, but there was no effect on liver glycogen at rest or after exercise. Feeding of the four levels of dietary fat in the form of soybean oil had no adverse effects and proved a safe and efficient method of providing concentrated energy to working horses.