The effects of an injectable anesthesia with 0.05 mg/kg medetomidine, 5 mg/kg ketamine, and 0.5 mg/kg butorphanol administered together intramuscularly were evaluated in 22 captive Humboldt penguins (Spheniscus humboldti, 10 male and 12 female), with a mean age of 8.5 ± 8.23 years. The birds fasted for18–24 hours prior to the procedure. Induction was followed by 4 distinct progressive responses of the birds to the anesthetic effect, including onset of initial effects at 2.0 ± 1.7 minutes (x̄ ± SD), sternal recumbency with the head still elevated at 2.2 ± 1.6 minutes, lowering and placing the beak tip to the ground at 3.6 ± 3.4 minutes, and lateral positioning of the neck and head at 4.2 ± 3.4 minutes. A general state of sedation, muscle relaxation, and analgesia were noted 10.0 ± 2.8 minutes postinjection. However, according to an established scoring system for the assessment of anesthetic depth in avian patients, a surgical plane of anesthesia was not achieved. Muscle relaxation determined by the same scoring system lasted for 31.4 ± 17.1 minutes. The penguins' mean respiratory rate did not demonstrate significant change and spontaneous ventilation was present throughout the procedure. Relative peripheral arterial oxygen saturation decreased significantly from 92.83 ± 5.77% at 10 minutes to 90.91 ± 5.77% at 40 minutes following induction. The birds' heart rate also decreased significantly from 112.55 ± 23.97 beats/min at 10 minutes to 101.65 ± 25.42 beats/min at 40 minutes. The measured cloacal temperatures were maintained within normal range despite ambient temperatures of up to 28.3°C (82.9°F). Reversal of medetomidine with 0.25 mg/kg atipamezole was conducted after 45.1 ± 7.3 minutes. Recovery was smooth but of variable duration with patients being able or willing to stand steadily in an upright position after 50.1 ± 34.6 minutes. One penguin died during recovery from a ruptured left ventricle and consecutive pericardial tamponade, but no predisposing factors were identified. The anesthetic protocol proved to be effective for noninvasive and minor painful procedures (eg, diagnostic imaging, blood collection). Disadvantages to the administration of the combined anesthetic agents in the penguins included a short period of muscle relaxation and smooth but potentially prolonged recovery. The safety of the anesthetic protocol described for Humboldt penguins in this report has to be evaluated critically against the the death of 1 penguin during recovery.