We examined the relationship in horses between osmotic fragility of erythrocytes (OFE) measured as the red cell haemolysis rates (HLR) in 0.56% NaCl and certain physiological characters. Animals were exercised on a treadmill: warmed up at 30% V̇O2max for 5 min or at 105% V̇O2max for 1 min, given an exercise test at 80 or 105% V̇O2max until fatigued, and then cooled down. The pH and temperature of the blood had a significant correlation for the HLR: r = 0.93 and 0.92 (P<.01) on arterial and mixed venous blood, respectively. In multiple regression analysis, heart rate and packed cell volume showed lower coefficients for the HLR. The present study suggested that, in exercising horses, 1) the increase of blood flow and release of erythrocytes from the spleen into the circulation have little effect on changes of OFE; 2) the decrease of blood pH promotes OFE; 3) the increase of the pH causes osmotic resistance in the cells and 4) an increase of blood temperature during exercise is associated with the decrease in the OFE.