Echocardiography represents the single most important tool for the evaluation of mammalian cardiac structure and function. Transthoracic echocardiography in dolphins has been limited by the animal's relatively large sternum and circumferential lung, both which interfere with effective sonographic imaging. Early attempts at transesophageal echocardiography (TEE) with sedated dolphins out of the water (Renner et.al 2001) were limited due to complications with sedation early in the study. The goal of this study was to develop a safe, effective and reproducible echocardiographic approach to evaluate the heart of the bottlenose dolphin (Tursiops truncatus). A TEE ultrasound probe was utilized, with the animal in the water as a trained medical behavior. The dolphin family at Dolphin Quest Oahu has had a considerable amount of training with voluntary gastroscopy, thus passing the esophageal probe and scanning animals voluntarily was not difficult. Good sonographic windows were only achieved after a forceful exhalation. Therefore the dolphins were conditioned to hold their breath following a forced exhalation during TEE. Over 100 TEE examinations were completed on 4 animals. Individual exams lasted up to 4 minutes with breath holds lasting up to 1 minute. TEE was performed with the dolphins upright and vertical in the water, and the sonographer and training staff dockside. The probe was advanced into the esophagus, typically to 60-70 cm. Rotating the probe allowed excellent visualization of all four chambers of the heart in both transverse and sagittal planes. High quality, reproducible images of the entire heart (mitral, tricuspid, aortic, pulmonary valves, atrial and ventricular septae, left and right ventricles, ascending aorta, and pulmonary artery) were obtained consistently using transesophageal imaging in conjunction with the behavioral breath hold following forceful exhalation. Color Doppler mapping demonstrated mild tricuspid regurgitation in all dolphins. Mild aortic regurgitation was noted in one dolphin that had a pedunculated mass arising from the sinotubular junction just above the aortic valve. This approach, and the normative echocardiographic data generated from this work, lays the foundation for future echocardiographic studies of cetaceans.