Objective- To evaluate physical methods for inducing death during the slaughter of American alligators (Alligator mississippiensis). Animals- 24 captive hatched-and-reared American alligators. Procedures- Baseline electroencephalograms (EEGs) were obtained for awake and anesthetized alligators. Corneal reflex, spontaneous blinking, and EEGs were evaluated after severance of the spinal cord, severance of the spinal cord followed by pithing of the brain, application of a penetrating captive bolt, or application of a nonpenetrating captive bolt (6 alligators/group). Results- Overall, alligators subjected to spinal cord severance alone differed from those subjected to the other techniques. Spinal cord severance alone resulted in postprocedure EEG power values greater than those in anesthetized alligators, whereas the postprocedure EEG power values were isoelectric for the other 3 techniques. Corneal reflex and spontaneous blinking were absent in all alligators immediately after application of a penetrating or nonpenetrating captive bolt. One of 6 alligators had a corneal reflex up to 1 minute after pithing, but all others within that group had immediate cessation of reflexes after pithing. Mean time to loss of spontaneous blinking and corneal reflex for alligators subjected to spinal cord severance alone was 18 minutes (range, 2 to 37 minutes) and 54 minutes (range, 34 to 99 minutes), respectively. Conclusions and Clinical Relevance- Spinal cord severance followed by pithing of the brain and application of a penetrating or nonpenetrating captive bolt appeared to be humane and effective techniques for inducing death in American alligators, whereas spinal cord severance alone was not found to be an appropriate method.