The aim of this study was to investigate the effect of ketamine and midazolam on preventing the development of morphine tolerance and dependence and withdrawal syndrome in mice. In the present study different groups of mice were received morphine (50 mg/kg, s.c.), morphine (50 mg/kg, s.c.) + ketamine (25, 50, 75 mg/kg, i.p.) , morphine (50 mg/kg , s.c.) + midazolam (0.5, 1, 2 mg/kg, i.p.) , morphine ( 50 mg/kg, s.c.) + [Ketamine (50 mg/kg, ip) + midazolam (1 mg/kg)] once a day for four days. Tolerance was assessed by administration of morphine (9mg/kg, ip) on fifth day. Withdrawal symptoms were assessed by administration of naloxone (4 mg/kg, i.p.) 2 hour after co-administration of morphine either with ketamine or with midazolam groups. The results showed that pretreatment with ketamine or midazolam decreases the degree of tolerance and withdrawal symptoms. On the other hand, co-administration of ketamine and midazolam before morphine treatment significantly decreased the tolerance and dependence and withdrawal symptoms. Ketamine and midazolam alone or in combination could also prevent the development of morphine tolerance and withdrawal symptoms. These effects can be attributed to NMDA-antagonistic behavior of ketamine and GABA-agonist behavior of midazolam. Furthermore, co- administration of ketamine and midazolam may exert its effect through two pathways, significantly preventing the development of morphine tolerance and withdrawal symptoms.