We previously demonstrated that Copenhagen (COP) and DA inbred rat strains show a wide difference in a test for aerobic treadmill running that correlated positively with isolated cardiac function. The purpose of this study was to test adenosine production as a candidate intermediate phenotype that may explain part of the difference in running and cardiac performance in these genetic models for low and high aerobic capacity. Adenosine production was measured as the activity of soluble 5'-nucleotidase and membrane-bound ecto-5'-nucleotidase in the membrane pellet and supernatant fractions of left and right ventricular muscle and gracilis muscle taken from 10 DA and 10 COP rats. Ecto-5'-nucleotidase activity in the membrane pellet of hearts from both DA and COP accounted for the vast majority of the total tissue adenosine production (>90% in the left ventricle and >80% in the right ventricle). Ecto-5'-nucleotidase activity in the pellet fraction was significantly higher in the left (22.4%) and right (46.1%) ventricles of DA rats compared with COP rats, with no differences in total protein content. There were no significant differences between the strains for 5'-nucleotidase activity in the cardiac supernatant, the gracilis pellet, or the gracilis supernatant. These data support the hypothesis that an increase in cardiac adenosine production may contribute to the greater aerobic running capacity of the DA rats.