Neuroanatomical, electrophysiological and pharmacological studies have provided indirect evidence indicating that GABAergic neurons play a key role in opiate analgesia mediated by the midbrain periaqueductal gray (PAG) and ventromedial medulla. Although these studies suggest that systemic administration of opiates inhibits GABA release in the PAG, there have been no investigations to date that have directly examined this issue. The present study was thus designed to determine whether systemic morphine injection inhibits GABA release in the PAG of awake, freely moving rats using in vivo microdialysis and subsequent HPLC analysis. Extracellular levels of GABA, glutamate, aspartate, glycine, homocysteic acid and taurine were monitored with the microdialysis technique in either the lateral or medial portion of the ventrocaudal PAG in unanesthetized, unrestrained rats. Amino acid release was induced by infusing veratridine (75 μM, a sodium channel activator) directly through the dialysis probe. The effect of veratridine alone and the effect of veratridine in the presence of systemic morphine on the concentrations of amino acids in the PAG dialysate were determined. There were no significant differences in the basal concentrations of GABA, taurine, aspartate, glutamate, homocysteic acid and glycine between dialysates collected from the medial versus the lateral ventrocaudal PAG. Glycine, taurine and glutamate were present in the highest concentrations in dialysis samples both before and after treatment with veratridine, whereas GABA, homocysteic acid and aspartate were present in the lowest concentrations. Perfusion of veratridine into the ventrocaudal PAG resulted in significant elevation of all amino acids investigated. Except for taurine, no significant difference in veratridine-induced release between the lateral and medial PAG was observed. Tetrodotoxin (TTX) significantly blocked veratridine-induced release of GABA, aspartate, glutamate, glycine and taurine but not homocysteic acid. When rats were injected with morphine (10 mg/kg i.p.), veratridine-induced release of GABA was selectively and significantly decreased in the lateral but not the medial PAG as compared to control rats injected with saline followed by veratridine perfusion. Systemic injection of morphine or saline caused no significant change in the basal concentration of amino acids in PAG dialysate samples. These findings are consistent with the proposed mechanism of action of morphine in the lateral ventrocaudal PAG and offer the first direct evidence that systemic opiates decrease GABA release in this midbrain region.