We compared electrocardiographic signals in hatchery-reared, non-spinally-transected, immature rainbow trout (Oncorhynchus mykiss Walbaum) under clove oil (25 ppm), tricaine methanesulfonate (tricaine, 60 ppm), and benzocaine (108 ppm) general anesthesia (35 min, 14 degrees C). For all 3 anesthetics, the mean heart rate (HR) and QRS amplitude did not differ, and QRS duration and QT interval were independent of HR. Heart rate variability (HRV) was significantly (4-fold, P=0.032) higher under benzocaine than under clove oil and tricaine, but did not differ between clove oil and tricaine. QRS duration differed between groups (P<0.001, F=121); benzocaine anesthesia resulted in longer QRS complexes compared to clove oil (P<0.001) and tricaine (P<0.001) anesthesia, and QRS complexes under clove oil were longer than those under tricaine (P<0.001). High HRV and QRS amplitude variation with benzocaine were associated with HR oscillations as anesthetic exposure time increased, and suggest benzocaine toxicity which may influence cardiac function studies. Similar clove oil and tricaine ECG patterns suggest comparable autonomic effects, and maintenance of myocardial excitability. Given its low cost, ease of use, and similar ECG profiles to tricaine, clove oil is a viable alternative for studies of cardiac function in anesthetized rainbow trout.