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The anesthetic effects of alfaxalone combined with medetomidine and butorphanol were investigated for ICR, BALB/c, and C57BL/6 mice. Mice were administered a combination of 0.5 or 0.75 mg/kg medetomidine and 5 mg/kg butorphanol with 30 or 40 mg/kg alfaxalone (0.5MBA30, 0.75MBA30 and 0.75MBA40, respectively). The drug combinations were administered subcutaneously and were compared with a widely used combination of 0.3 mg/kg medetomidine, 4 mg/kg midazolam, and 5 mg/kg butorphanol (MMB). All three MBA combinations achieved surgical anesthesia, although the recovery time was longer with 0.75MBA30 and 0.75MBA40 compared with 0.5MBA30. Furthermore, several mice exhibited a considerable jumping reaction immediately after injection with 0.75MBA30 and 0.75MBA40. Therefore, 0.5MBA30 may be suitable for inducing surgical anesthesia in the mouse strains tested. The anesthetic scores for 0.5MBA30 were improved compared with those of MMB in all three mouse strains; however, the body temperature drop in C57BL/6 mice was greater with 0.5MBA30. Our results show that the alfaxalone combination, 0.5MBA30, should allow surgical operations that are more stable in more strains of mice than MMB, although the combination may cause hypothermia, especially in C57BL/6 mice.