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In this study, we investigated a method for accurately measuring myocardial T1 for the quantification of myocardial blood flow (MBF) with arterial spin labeling (ASL). A single-shot gradient-echo (GE)-based ASL sequence with an adiabatic hyperbolic secant inversion recovery pulse was modified to acquire a pair of myocardial T1's within a breath-hold. A multivariable regression algorithm that accounted for the magnetization saturation effects was developed to calculate T1. The MBF was then determined with a well-developed model. The accuracy of our T1 calculation was first evaluated in a phantom, and then in six dogs for the MBF calculation, with ( N = 4) and without ( N = 2) coronary artery stenosis. In the phantom study, the accuracy of T1 measured with a slice-selective inversion prepared pulse was within 2.5% of error. In healthy dogs, the MBF increased 2-5 times during vasodilation. In contrast, regional differences of MBF were well visualized in the stenotic dogs during vasodilation (perfusion reserve of 2.75 ± 0.83 in normal myocardium, and 1.46 ± 0.75 in the stenotic area). A correlation analysis revealed a close agreement in MBF between the ASL and microsphere (MS) in both healthy and stenotic dogs. In summary, the modified ASL technique and T1 regression algorithm proposed here provide an accurate measurement of myocardial T1 and demonstrate potential for reliably assessing MBF at steady state. Magn Reson Med 53:1135-1142, 2005. © 2005 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]