The objective of this study was to determine the effects of 10 consecutive days of moderate intensity training on 1) the concentration of middle gluteal muscle Na(+)-K(+)-ATPase as determined by vanadate-facilitated 3H[ouabain binding; and 2) plasma potassium regulation before, during and after exercise at 100% of the pre-training maximum rate of oxygen consumption (VO2max). Six mature, unfit Thoroughbred horses completed both incremental (for determination of VO2max) and high-intensity exercise protocols before (HI1) and after (HI2) training. There additional horses undertook no training or exercise tests and served as controls for determination of middle gluteal muscle Na(+)-K(+)-ATPase concentration. Training consisted of 10 consecutive days of running at 55% VO2max for 60 min per day (13-14 km/day). For each high intensity exercise protocol, horses completed a 10 min warm-up at 50% VO2max, followed by exercise at 100% of pre-training VO2max (6 degrees incline, mean speed 9.8 m/s) until fatigue. Training resulted in a 13.8% increase in resting plasma volume (pre: 20.9 +/- 0.8 l; post: 23.8 +/- 0.9 l; P = 0.03), and an 8.9% increase in VO2max (pre: 142 +/- 4 ml/kg/min; post: 155 +/- 4 ml/kg/min; P = 0.004) during HI. Peak values for plasma potassium concentration and content during exercise decreased by 13% (P = 0.02) and 7% (P = 0.0002), respectively, after training whereas training had no effect on increases in packed cell volume, plasma total solids, and erythrocyte K+ concentration and content during exercise. Following training, there was also a significant (23%) increase in Na(+)-K(+)-ATPase concentration in biopsies of middle gluteal muscle, as measured by vanadate-facilitated 3H[ouabain binding. We conclude that 10 days of moderate intensity exercise results in increases in skeletal muscle Na(+)-K(+)-ATPase and attenuation in the elevation in plasma K+ during high intensity exercise at the same absolute workload. The increase in middle gluteal muscle Na(+)-K(+)-ATPase concentration is consistent with decreases in K+ efflux from working muscle during exercise.