Recently, a mixture of medetomidine, midazolam and butorphanol (MMB) has been used as an injectable general anesthetic agent for laboratory animals. The purpose of this study was to establish data to encourage practical usage of MMB, and to clarify the effects of MMB on the respiratory function in rats. To compare the anesthetic efficacy between the injection routes, the anesthetic effects of MMB by subcutaneous (s.c.) or intraperitoneal (i.p.) injection were evaluated in rats. To assess the respiratory function, the blood gas parameters and electrolytes were assessed in serial venous blood samples collected from before s.c. injection of MMB to 270 min after the injection. Recovery from anesthesia and the respiratory changes after atipamezole injection at 30 min after MMB injection was also examined. Subcutaneous injection of MMB was associated with more rapid induction and a longer duration of anesthesia as compared to i.p. injection. The blood gas analysis findings showed MMB had effects on respiratory function, that is, elevations of the partial pressures of carbon dioxide and bicarbonate and reduction of the blood pH. Atipamezole injection resulted in recovery from the MMB-induced anesthetic effect as well as respiratory depression. In conclusion, MMB provides more effective anesthesia administered by s.c. injection compared to i.p. injection and induces respiratory change. These changes were counteracted by atipamezole. Therefore, we recommend MMB administered by s.c. injection for anesthesia, followed by injection of atipamezole after the operative procedure to allow recovery.