The combined effects of endurance run training and renal hypertension on cytosolic Ca²⁺ concentration ([Ca²⁺]_c) dynamics and Na⁺-dependent Ca²⁺ regulation in rat left ventricular cardiomyocytes were examined. Male Fischer 344 rats underwent stenosis of the left renal artery [hypertensive (Ht), n = 18] or a sham operation [normotensive (Nt),n = 20]. One-half of the rats from each group were treadmill trained for >16 wk. Cardiomyocyte fura 2 fluorescence ratio transients were recorded for 7 min during electrical pacing at 0.5 Hz, 2 mM extracellular Ca²⁺ concentration, and 29°C. The rate of [Ca²⁺]_c decline was not changed by run training in the Nt group but was reduced in the Ht group. At 7 min, cardiomyocytes were exposed to 10 mM caffeine in the absence of Na⁺ and Ca²⁺, which triggered sarcoplasmic reticular Ca²⁺ release and suppressed Ca²⁺efflux via Na⁺/Ca²⁺ exchanger. External Na⁺ was then added, and Na⁺-dependent Ca²⁺ efflux rate was recorded. Treadmill training significantly enhanced Na⁺-dependent Ca²⁺efflux rate under these conditions in the Nt group but not in the Ht group. These data provide evidence that renal hypertension prevents the normal run training-induced modifications in diastolic [Ca²⁺]_c regulation mechanisms, including Na⁺/Ca²⁺ exchanger.