The heart critically depends on a continuous supply of oxygen, provided by myocardial blood flow (MBF), but reliable quantification of MBF during maximal exercise is not feasible in humans. We measured MBF at rest and during similar absolute (150 W) and relative (70% of ) cycling exercise workloads in seven healthy untrained (UT) and seven healthy endurance-trained (ET) men by positron emission tomography and [15O]H2O-tracer. The maximum MBF was extrapolated using the linear relationship between cardiac work load and MBF. MBF was also measured during intravenous infusion of standard high dose adenosine known to cause maximal vasodilation in the myocardial vasculature. Maximal exercise-induced MBF was calculated to be 5.0 ± 1.5 mL/g/min in UT and 4.5 ± 1.7 mL/g/min in ET (p = 0.6), which was 6.9-fold and 6.5-fold higher from their respective resting values. Adenosine-induced MBF was 3.9 ± 1.4 mL/g/min in UT and 2.3 ± 0.9 mL/g/min in ET, being significantly lower than maximal exercise-induced MBF in both groups. In addition, MBF was lower in the ET than in the UT group in both conditions. Myocardial vascular resistance responses and conclusions depended on whether the estimated maximal exercise calculations were based on mean arterial or systolic blood pressure estimations. This study thus demonstrates that estimated maximal MBF during exercise exceeds that achieved with adenosine infusion, and its increase from rest in humans may be higher than previously considered. Endurance training appears, however, not to increase peak MBF per gram of myocardium.