A safe and effective technique for underwater anesthesia of green sea turtles (Chelonia mydas) was developed to allow fully submerged in-water measurements of auditory evoked potentials (AEP) without myogenic artifact. Turtles were anesthetized using medetomidine 50 μg/kg and ketamine 5 mg/kg IV and ventilated via a custom-designed double-cuffed extended endotracheal tube while submerged to an ear depth of 10 cm for up to 60 min. Procedures conducted on manually restrained turtles, submerged within range of the water surface so they could breathe voluntarily, were compared with those conducted using anesthesia, based on sea turtle venous blood gas measurements and the ability to record AEPs. Quality AEPs were recorded from both anesthetized turtles and four of seven manually restrained turtles, whereas AEP recordings were impossible for the remaining manually restrained turtles because of myogenic artifact. Manual restraint was superior to anesthesia for turtles that did not resist restraint (better venous blood oxygenation, acceptable AEPs), but anesthesia was superior to manual restraint when compared with turtles that did resist (marked lactic acidosis, and AEPs not possible). Underwater anesthesia of sea turtles is a viable option for specialized physiologic testing requiring submerged inactive subjects.