Tail-tip biopsy for genotyping of genetically modified mice older than 21 d typically is performed by using isoflurane anesthesia. Isoflurane-induced changes in behavior and metabolism can result in unexpected complications and death. We investigated whether cryoanalgesia by using ethylene chloride spray would be an effective local anesthetic for tail-tip biopsies in mice. C57BL/6J mice were allocated randomly into 4 groups (n = 10 each) to receive isoflurane anesthesia with tail biopsy, ethylene chloride spray on the tip of the tail before biopsy, ethylene chloride spray without biopsy, or no treatment. Blood glucose was measured periodically in both groups undergoing tail biopsy, and the tail-pinch assay was performed in all mice that received ethylene chloride spray. Body weight, water, and food intake were measured daily for 2 wk. In both groups undergoing tail biopsy, blood glucose levels at 15 min were significantly higher than those after 2 min. This elevation was greater and more prolonged after 30 min in mice that received isoflurane compared with ethylene chloride spray. Tail-pinch latency at 20 min was greater than that after 2 min in all mice that received ethylene chloride spray. All mice gained weight, and there was no difference in food and water intake among groups. We conclude that ethylene chloride spray is an effective local anesthetic and a valuable alternative to isoflurane.