In this report we describe a means of general anesthesia for medium-duration (i.e., 20 to 60 min) surgery of infant mice. We tested isoflurane inhalation (2.0% isoflurane in air or oxygen during induction, and 1.5% after surgical anesthesia) anesthesia of 6-to 10-day-old C57BL/6JJcl mice and obtained safe, effective, and reproducible results.