During short-term maximal exercise, horses have impaired pulmonary gas exchange, manifested by diffusion limitation and arterial hypoxemia, without marked ventilation-perfusion (A/) inequality. Whether gas exchange deteriorates progressively during prolonged submaximal exercise has not been investigated. Six thoroughbred horses performed treadmill exercise at ~60% of maximal oxygen uptake until exhaustion (28-39 min). Multiple inert gas, blood-gas, hemodynamic, metabolic rate, and ventilatory data were obtained at rest and 5-min intervals during exercise. Oxygen uptake, cardiac output, and alveolar-arterial PO₂ gradient were unchanged after the first 5 min of exercise. Alveolar ventilation increased progressively during exercise, from increased tidal volume and respiratory frequency, resulting in an increase in arterial PO₂ and decrease in arterial PCO₂. At rest there was minimal A/ inequality, log SD of the perfusion distribution (log SD) = 0.20. This doubled by 5 min of exercise (log SD = 0.40) but did not increase further. There was no evidence of alveolar-end-capillary diffusion limitation during exercise. However, there was evidence for gas-phase diffusion limitation at all time points, and enflurane was preferentially overretained. Horses maintain excellent pulmonary gas exchange during exhaustive, submaximal exercise. Although A/ inequality is greater than at rest, it is less than observed in most mammals and the effect on gas exchange is minimal.