This study aims to quantify subendocardial and subepicardial myocardial blood flow (MBF) from dynamic contrast-enhanced MRI and to compare semi-quantitative and quantitative analysis methods. 17 healthy volunteers (9 males, mean age 34 ± 8) were scanned during adenosine stress and at rest. A “semi-quantitative” myocardial perfusion index (MPI) was calculated based on maximal upslopes of signal intensity-time profiles for a mid-ventricular myocardial slice. In addition, absolute MBF (ml/g/min) was estimated using Fermi-constrained deconvolution. On semi-quantitative analysis, the ratio of subendocardial to subepicardial MPI was 0.98 ± 0.1 at stress and 1.16 ± 0.09 at rest, P < 0.0001. The MPRI (i.e. the ratio of stress over rest MPI) for the subendocardium was 1.54 ± 0.3 versus 1.81 ± 0.35 for the subepicardium, P = 0.03. For quantitative analysis, the ratio of subendocardial to subepicardial MBF was 0.91 ± 0.11 at stress versus 1.17 ± 0.16 at rest, P < 0.0001. The subendocardial MBF reserve was also lower than in the subepicardium (2.6 ± 0.75 vs. 3.32 ± 0.93, P = 0.027). In conclusion, semi-quantitative and quantitative analysis of dynamic contrast-enhanced MRI shows higher subendocardial blood flow at rest and reduced subendocardial perfusion reserve compared to the subepicardium.