We have previously reported that both skeletal muscle receptor and arterial baroreceptor afferent inputs activate neurons in the dorsolateral (DL) and lateral (L) regions of the midbrain periaqueductal gray (PAG). In this study, we determined if the excitatory amino acid, glutamate (Glu), is released to mediate this increased activity in these regions. Static contraction of the triceps surae muscle for 4 minutes was evoked by electrical stimulation of the L7 and S1 ventral roots in cats. Activation of arterial baroreceptor was induced by intravenous injection of phenylephrine (PE). Endogenous release of Glu from the PAG was recovered by a microdialysis probe. Glu concentration was measured by the HPLC method. Muscle contraction increased mean arterial pressure (MAP) from 98±10 to 149±12 mm Hg (p<0.05) and increased Glu release in the DL and L regions of the middle PAG from 0.39±0.10 to 0.73±0.12 µM (87%, p<0.05) in intact cats. After sinoaortic denervation and vagotomy, contraction increased MAP from 95±12 to 158±15 mm Hg, and Glu from 0.34±0.08 to 0.54±0.10 µM (59 %, p<0.05). The increases in arterial pressure and Glu were abolished by muscle paralysis. PE increased MAP from 100±13 to 162±22 mm Hg, and Glu from 0.36±0.10 to 0.59±0.18 µM (64 %, p<0.05) in intact animals. Denervation abolished this Glu increase. Summation of the changes in Glu evoked by muscle receptor and arterial baroreceptor afferent inputs was greater than the increase in Glu produced when both reflexes were activated simultaneously in intact state (123 % vs 87 %). These data demonstrate that: 1) activation of skeletal muscle receptors evokes release of Glu in the DL and L regions of the middle PAG; and 2) convergence of afferent inputs from muscle receptors and arterial baroreceptors in these regions inhibits the release of Glu. These results suggest that the PAG is a neural integrating site for the interaction between the exercise pressor reflex and the arterial baroreceptor reflex.