Introduction: A MR imaging (MRI) method has been developed to determine quantitatively myocardial perfusion (P) in the rat heart in vivo. This method has the potential to non-invasively measure cardiac perfusion without the use of a contrast agent by exploiting the endogenous contrast from flowing blood itself. Method and Results: Principle of the technique is the arterial spin labeling of endogenous water protons within the short axis imaging slice. Arterial spin labeling techniques are based on a model that uses inflow effects to relate intrinsic changes in longitudinal relaxation (T-1) to tissue perfusion. Perfusion is determined from the difference between a slice selective and a global inversion recovery experiment. Perfusion was determined at rest and during hyperemia induced by intravenous adenosine (3 mg/(kgmin)). The MR perfusion values were compared with perfusion data obtained in the same animal using the colored microspheres (MS) technique as the gold standard. The MR perfusion (mean +/- SEM) was 3.3 +/- 0.2 ml/min/g at rest and 4.6 +/- 0.6 ml/min/g during adenosine. Perfusion values obtained by colored MS were 3.4 +/- 0.2 and 4.7 +/- 0.8 ml/min/g at rest and during vasodilation, respectively. Adenosine decreased mean arterial pressure (MAP) from 120 to 65 mmHg which implies a reduction of coronary resistance (CR) to about 50% of baseline. Conclusion: Our study shows that quantitative mapping of perfusion may be performed non-invasively by MRI. The MR perfusion data are in excellent correlation with data obtained by the well-established colored MS technique. Determination of perfusion reserve confirms that coronary perfusion is highly dependent on blood pressure due to changes in CR.