Objective To examine the effects of different doses of midazelam on ED50 of emulsified isoflurane and to determine the type of interaction between them for hypnosis by isobelographic analysis in rats. Methods One hundred and twenty-five adult male SD rats weighing 240-300 g were randomized into 5 groups (n=25 each): group Ⅰmidazolam (M); group Ⅱ emulsified isnflurane (Ⅰ); group Ⅲ ,Ⅳ ,Ⅴ, 1/4, 1/2 and 3/4 ED50 of midazelam for hypnosis + emulsified isoflurane (MI1 , MI2, MI3). Up-and-down sequential experiment was used to determine ED50of midazolam and emulsified isoflurane for loss of righting reflex in group Ⅰ -Ⅴ . The intial dose of midazolm was 17.3 mg/kg in group M. The initial doses of emulsified isoflurane (120 mg/ml) were 0.55 (in group Ⅰ, 0.22 (MI1), 0.19 (MI2) and 0.12 ml/kg (MI3) respectively. In group MI1-3 midazolam was injected over 15 seconds and after an interval of 2.5 min emulsified isoflurane was injected over 10 s. ED50 was calculated using Dixon-Mood method. Isebolographic and algebraic analyses were used to determine the type of interaction between midazolam and emulsified isoflurane. Results The five groups were comparable with respect to M/F sex ratio and body weight. The Edso of midazolam was 26.0 mg/kg in group M. Midazolam 6.5, 13.0 and 19.5 mg/kg were given in group MI1 , MI2 and MI3 respectively. The ED50 of emulsified isoflurane was 0.67 (ingroup Ⅰ), 0.30 (MI1), 0.22 (MI2) and 0.18 ml/kg (MI3) respectively. The isobolographic analysis indicated that with increasing doses of midazolam, the Edw of emulsified isoflurane decreased progressively in a non-linear fashion. The isobolographic and algebraic analyses demonstrated that the interaction between midazolam and emulsified isoflurane was synergistic for hypnosis. Conclusion The hypnosis is synergistic when midazolam 6.5,13 mg/kg are combined with emulsified isoflurane and additive when midazolam 19.5 mg/kg is combined with emulsified isoflurane.