Recent clinical studies using a non-fluoroscopic three-dimensional (3D) left ventricular (LV) mapping system suggested reduced endocardial voltage amplitudes measured in zones sustaining myocardial ischemia or infarction. However, the direct relationship between myocardial perfusion and endomyocardial voltage amplitudes has not been fully elucidated. In a pig model of chronic myocardial ischemia (n = 20), LV endocardial unipolar voltage (UpV) mapping was performed using the Biosense 3D navigation system (Johnson and Johnson, Warren, New Jersey, USA) 4 weeks after ameroid constrictor placement around the left circumflex coronary artery. Echocardiography was used to assess regional changes in myocardial wall thickening (MT) and fluorescent microspheres (4 x 10/injection) were used to quantify rest regional myocardial blood flow (MBF) in ischemic (left circumflex) and remote non-ischemic (left anterior descending) regions. UpV measurements were reduced in ischemic compared to non-ischemic zones (9.9+/-3.3 compared with 13.3+/-3.3 mV, P = 0.03). This corresponded to changes in endocardial MBF and MT, which were both noted to be significantly reduced in the ischemic compared to the non-ischemic area (MBF, 0.50+/-0.16 compared with 0.74+/-0.15 ml/g per min, P = 0.001; MT, 26.1+/-12.0 compared with 37.4+/-10.1%, P=0.003). A positive linear correlation was found between UpV at rest and endomyocardial (but not epicardial) perfusion: UpV (mV) = 7.8+5.9xMBFendocardial (r = 0.32, P = 0.05). Chronic myocardial ischemia, resulting in reduced perfusion and function at rest (that is, hibernating myocardium), is characterized by a significant reduction ( approximately 25%) in endocardial UpV potentials, which correlates with reduced endomyocardial blood flow and tissue perfusion at rest.