The present study reports on a new calibration of the magnetic resonance imaging (MRI) signal intensity of a fast gradient-echo sequence used for in vivo myocardial perfusion quantification in patients. The signal from a FAST sequence preceded by a arrhythmia-insensitive magnetization preparation was calibrated in vitro using tubes filled with various gadolinium (Gd) solutions. Single short-axis views of the heart were obtained in patients (n = 10) with normal cardiac function. Myocardial and blood signal intensity were converted to concentration of Gd according to the in vitro calibration curve and fitted by a one-compartment model. K₁ [first-order transfer constant from the blood to the myocardium for the gadolinium-diethylene-triamine-pentaacetic acid (Gd-DTPA)] and V_d (distribution volume of Gd-DTPA in myocardium) obtained from the fit of the MRI-derived perfusion curves were 0.72 ± 0.22 (mL/min/g) and 15.3 ± 5.22%. These results were in agreement with previous observations on animals and demonstrated that a reliable measurement of myocardial perfusion can be obtained by MRI in patients with an in vitro calibration procedure.