The aim of the present study was to evaluate the Ca2+ handling and contractility properties of cardiomyocytes isolated from rats with high intrinsic aerobic exercise capacity. Standard-performance (SP) and high-performance (HP) rats were categorized with a treadmill progressive exercise test according to the exercise time to fatigue (TTF). The SP group included rats with TTF between 16.63 and 46.57 min, and the HP group included rats with TTF>46.57 min. Isolated ventricular cardiomyocytes were dissociated from the hearts of SP and HP rats, and intracellular global Ca2+ ([Ca2+]i) transients were measured. The [Ca2+]i transient peak was increased in the HP group relative to the SP group (5.54+/-0.31 vs. 4.18+/-0.12 F/F0; P<or=0.05) and was positively correlated with the TTF attained during the progressive test (r=0.81). We also performed contractility measurements in isolated cardiomyocytes and found higher amplitude of contraction in the HP group compared with the SP group (6.7+/-0.2 vs. 6.0+/-0.3% resting cell length; P<or=0.05). To reinforce the intrinsic differences between SP and HP rats, we performed Western blot experiments and observed increased expression of sarco(endo)plasmic reticulum Ca2+-ATPase type 2a (1.30+/-0.07 vs. 1.74+/-0.18 arbitrary units; P<or=0.05) and ryanodine receptor type 2 (1.86+/-0.13 vs. 3.57+/-0.12 arbitrary units; P<or=0.05) in HP rats. In summary, our data showed important intrinsic differences in cardiomyocyte properties that could explain some of the divergence observed in rats with high intrinsic aerobic exercise capacity.