OBJECTIVE This study evaluated variables relevant to creating myocardial lesions using high-intensity focused ultrasound (HIFU). Without an effective means of tracking heart motion, lesion formation in the moving ventricle can be accomplished by intermittent delivery of HIFU energy synchronized by electrocardiographic triggering. In anticipation of future clinical applications, multiple lesions were created by brief HIFU pulses in calf myocardial tissue ex vivo. METHODS Experiments used f-number 1.1 spherical cap HIFU transducers operating near 5 MHz with in situ spatial average intensities of 13 and 7.4 kW/cm2 at corresponding depths of 10 and 25 mm in the tissue. The distance from the HIFU transducer to the tissue surface was measured with a 7.5-MHz A-mode transducer coaxial and confocal with the HIFU transducer. After exposures, fresh, unstained tissue was dissected to measure visible lesion length and width. Lesion dimensions were plotted as functions of pulse parameters, cardiac structure, tissue temperature, and focal depth. RESULTS Lesion size in ex vivo tissue depended strongly on the total exposure time but did not depend strongly on pulse duration. Lesion width depended strongly on the pulse-to-pulse interval, and lesion width and length depended strongly on the initial tissue temperature. CONCLUSIONS High-intensity focused ultrasound creates well-demarcated lesions in ex vivo cardiac muscle without damaging intervening or distal tissue. These initial studies suggest that HIFU offers an effective, noninvasive method for ablating myocardial tissues to treat several important cardiac diseases.