Previous studies have demonstrated that electroacupuncture (EA) attenuates sympathoexcitatory reflex responses by activating a long-loop pathway involving the hypothalamic arcuate nucleus (ARC), midbrain ventrolateral periaqueductal gray (vlPAG) and rostral ventrolateral medulla (rVLM). Neurons in the ARC provide excitatory input to the vlPAG, while the vlPAG inhibits neuronal activity in the rVLM. -aminobutyric acid (GABA) and glutamate (Glu) have been identified in the vlPAG. Endocannabinoids (ECs), acting as atypical neurotransmitters, inhibit the release of both neurotransmitters in the hypothalamus and midbrain through a presynaptic cannabinoid CB₁ receptor mechanism. The EC system has been observed in the dorsal but not in the vlPAG. Since it is uncertain if ECs influence GABA and Glu in the vlPAG, we hypothesized that EA modulates these neurotransmitters release in the vlPAG through a presynaptic cannabinoid CB₁ receptor mechanism. We measured the release of GABA and Glu simultaneously by using HPLC to assess samples collected with microdialysis probes inserted unilaterally into the vlPAG of intact anesthetized rats. Twenty eight min of EA (2 Hz, 2-4 mA, 0.5 ms) at P5-6 acupoints reduced the release of GABA by 39% during and 44% 15 min after EA. Thirty five min after EA, GABA concentrations returned to pre-EA levels. In contrast, sham EA did not change the vlPAG GABA concentration. Blockade of CB₁ receptors with AM251, a selective receptor antagonist, reversed the EA-modulated changes in GABA concentration, while microperfusion of vehicle into the vlPAG did not alter EA-modulated GABA changes. Additionally, we observed no changes in the vlPAG Glu concentrations during EA, although the Glu baseline concentration was much higher than GABA (3541 ± 373 vs. 33.8 ±8.7 nM, Glu vs. GABA). These results suggest that EA modulates sympathoexcitatory reflex responses by decreasing the release of GABA, but not Glu, in the vlPAG through a presynaptic cannabinoid CB₁ receptor mechanism.