Sex differences are observed in the development of tolerance to antinociceptive effect of opioid drugs such as morphine, but the underlying Mechanisms remain unclear. Critical role of glutamate in the development and maintenance of opioid tolerance has been reported by many investigators. There are also evidences about interaction between gonadal hormones and neuromodulatory systems including opioidergic and glutamatergic systems, the present study, examined the sex differences on the glutamate level in the nucleus accumbens in morphine tolerant rats using in vivo microdialysis. Rats received morphine sulfate (7 mg/kg, s.c.) for 8 consecutive days, for tolerance induction and nociceptive responsiveness was assessed by the tail-flick. In microdialysis study, a microdialysis probe was implanted into the left nucleus accumbens core of rats and CSF (cerebrospinal fluid) dialysates were collected. The concentration of glutamate was measured by high performance liquid chromatography with a fluorescence detector. The results showed that after chronic morphine administration, antinociceptive tolerance in male rats was significantly greater than females (P<0.05). Sex differences in morphine tolerance disappeared with gonadectomy of animals. There was also significant sex difference in glutamate level in nucleus accumbens of morphine tolerant rats (P<0.05), Glutamate level was decreased after ovariectomy of female rats (P<0.05), but gonadectomy had not significant effect on glutamate level in males. The result of this study, provide evidences of sex differences in development of tolerance to morphine in rats and mediatory roles of gonadal hormones and glutamate concentration in these differences.