Electrical stimulation in the periaqueductal gray (PAG) can inhibit dorsal horn cell responses to both innocuous and noxious cutaneous stimuli. This inhibition is believed to be due to the release of serotonin (5-HT) into the dorsal horn of the spinal cord from descending axons of the nucleus raphe magnus and the adjacent reticular formation. It is still not clearly known which subtypes of 5-HT receptors are involved in the PAG-induced inhibition. Extracellular single-unit recordings of dorsal horn cell activity, in combination with drug administration through a microdialysis fiber, were used to test the role of 5-HT3 receptors in PAG-induced inhibition. The responses of the cells to mechanical stimulation of the skin (BRUSH, PRESS and PINCH) and to the same stimuli while stimulating PAG were recorded. When the 5-HT3 antagonist, ondansetron, was perfused through the microdialysis fiber, not only was the background activity of the cell increased, but also the responses to BRUSH, PRESS and PINCH stimuli. The PAG-induced inhibition of responses to the same stimuli was partially or completely blocked by ondansetron. Another 5-HT3 antagonist, zacopride, did not increase the background activity or responses to PRESS and PINCH, yet this agent, like ondansetron, blocked PAG inhibition. The 5-HT3 agonist, phenylbiguanide, inhibited the background activity and the responses to mechanical stimuli. These results suggest that 5-HT released in the dorsal horn by stimulation in the PAG excites inhibitory interneurons through 5-HT3 receptors, resulting in inhibition of dorsal horn neurons.