Eight horses exercised to fatigue were used to characterize the resulting changes in blood pH, in blood lactate, free fatty acid, bicarbonate, and ammonia concentrations, and in muscle glycogen concentrations. The exercise test was conducted at a speed of 4.5 m/s on a motorized equine treadmill set at a 9% grade. At fatigue, all variables differed significantly (P less than 0.05) from base-line values. Heart rate averaged 191.1 +/- 6.5 beats/min at fatigue, and the plasma lactate concentrations increased from 7.8 +/- 0.95 mg/dl to 94.3 +/- 19.2 mg/dl. Ammonia concentrations increased from 66.7 +/- 6.9 mumol/L before exercise to 136.9 +/- 18.6 mumol/L at fatigue. Bicarbonate concentrations decreased from 31.3 +/- 0.4 mM to 21.1 +/- 1.8 mM, and pH decreased from 7.37 +/- 0.01 to 7.28 +/- 0.04. Free fatty acid concentrations were higher at fatigue and increased throughout the recovery period. Exercise resulted in a 25% decrease of muscle glycogen concentration in gluteus medius specimens.