The stimulation of the periaqueductal gray (PAG) produces behavioral analgesia in rats, cats, monkeys, and humans. This analgesia is believed to be mediated by several neurotransmitter systems, including the serotonergic, noradrenergic, glycinergic, γ-aminobutyric acidergic, and opiatergic systems. The present study was designed to determine whether PAG stimulation produces the release of serotonin (5-HT), norepinephrine (NE), Gly, and γ-aminobutyric acid in the spinal cord dorsal horn and whether the release of these neurotransmitters by PAG stimulation is associated with a long-lasting inhibition of the evoked nociceptive responses of dorsal horn neurons. The effect of different frequencies of stimuli on the release of neurotransmitters in the spinal cord was also examined. Microdialysis in combination with HPLC was used to measure the concentrations of neurotransmitters in the lumbar dorsal horn before, during, and after electrical stimulation of the PAG. The PAG was stimulated with electrical pulses at 333 Hz first and then at 67 Hz with the same intensity for 27 min, respectively. Both stimulus frequencies produced a significant increase in the release of 5-HT, NE, Gly, and Asp in the spinal dialysate, but the low-frequency stimulus was more potent in causing the release of neurotransmitters. Low-frequency stimulation also significantly increased the release of Glu. The time course of inhibition of dorsal horn neurons induced by long-lasting PAG stimulation corresponded to the time course of neurotransmitter release. Therefore, the results suggest that the long-lasting inhibition induced by PAG stimulation is mediated in part by the release of 5-HT, NE, and inhibitory amino acids in the spinal cord.