Objective To investigate whether environmental cues associated with different properties of morphine could regulate the extracellular levels of glutamate and gamma-aminobutyric acid (GABA) in the hippocampal ventral subiculum, which play a critical role in the reinstatement of drug-seeking behavior induced by environmental cues. Methods Conditioning place preference (CPP) and conditioning place aversion (CPA) models were used to establish environment associated with rewarding and aversive properties of morphine respectively. Microdialysis and high performance liquid chromatography were used to measure the extracelluar level of glutamate and GABA in the ventral subiculum under these environmental cues. Results Exposure to the environmental cues associated with rewarding properties of morphine resulted in a decrease (approximately 11%) of extracellular level of GABA in ventral subiculum, and exposure to the environmental cues associated with aversive properties of morphine resulted in an increase (approximately 230%) of extracellular level of glutamate in ventral subiculum. Conclusion Environmental cues associated with different properties of morphine modulate the release of distinct neurotransmitters in the hippocampal ventral subiculum possibly through different neural circuit.