Although the safety and efficacy of tricaine methanesulfonate (MS222) for anesthesia of fish are well established, other used less commonly in fish have been less extensively evaluated. Therefore, we compared gradual cooling, lidocaine hydrochloride (300, 325, and 350 mg/L), metomidate hydrochloride (2, 4, 6, 8, and 10 mg/L), and isoflurane (0.5 mL/L) MS222 (150 mg/L) for anesthesia of adult zebrafish. The efficacy and safety of each agent was evaluated by observing loss equilibrium, slowing of opercular movement, response to tail-fin pinch, recovery time, and anesthesia-associated mortality rates. At 15 min after anesthetic recovery, we used a novel-tank test to evaluate whether anesthetic exposure influenced term anxiety-like behavior. Behavioral parameters measured included latency to enter and number of transitions to the half of the tank, number of erratic movements, and number of freezing bouts. Behavior after anesthesia was unaltered of the anesthetic used. Efficacy and safety differed among the anesthetics evaluated. Gradual cooling was useful short procedures requiring immobilization only, but all instrumentation and surfaces that come in contact with fish must maintained at approximately 10°C. MS222 and lidocaine hydrochloride at 325 mg/L were effective as anesthetic agents for procedures in adult zebrafish, but isoflurane and high-dose lidocaine hydrochloride were unsuitable as sole anesthetic due to high (30%) mortality rates. Although MS222 remains the best choice for generating a surgical plane of anesthesia, metomidate hydrochloride and gradual cooling were useful for sedation and immobilization for nonpainful procedures. Copyright 2014 by the American Association for Laboratory Animal Science.