Arterial spin labeling (ASL) provides noninvasive measurement of tissue blood flow, but sensitivity to motion has limited its application to imaging of myocardial blood flow. Although different cardiac phases can be synchronized using electrocardiography triggering, breath holding is generally required to minimize effects of respiratory motion during ASL scanning, which may be challenging in clinical populations. Here a freebreathing myocardial ASL technique with the potential for reliable clinical application is presented, by combining ASL with a navigator-gated, electrocardiography-triggered TrueFISP readout sequence. Dynamic myocardial perfusion signals were measured at multiple delay times that allowed simultaneous fitting of myocardial blood flow and arterial transit time. With the assist of a nonrigid motion correction program, the estimated mean myocardial blood flow was 1.00 6 0.55 mL/g/min with a mean transit time of~400 msec. The intraclass correlation coefficient of repeated scans was 0.89 with a mean within subject coefficient of variation of 22%. Perfusion response during mild to moderate stress was further measured. The capability for noninvasive, free-breathing assessment of myocardial blood flow using ASL may offer an alternative approach to first-pass perfusion MRI for clinical evaluation of patients with coronary artery disease. Magn Reson Med 64:1289-1295,