The purpose of the present study was to determine whether glutamate-induced mechanical sensitization of the masseter muscle in human volunteers involves activation of peripheral N-methyl-D-aspartate (NMDA) receptors. Healthy male volunteers (n=18) participated in this randomized, two-session study. During each session, the volunteers received two injections into the right masseter muscle. An initial injection of glutamate (1 M, 0.2 ml) alone was followed 30 min later by a second injection of glutamate alone or glutamate combined with ketamine (10 mM). Pressure pain threshold (PPT) was assessed over the right masseter muscle at and 2 cm above the injection site, as well as over the right temporalis muscle and left masseter muscle prior to the first injection. The PPT was reassessed at all four sites every 5 min from 10 to 30 min after the second injection and once again 60 min after the second injection. Glutamate-evoked muscle pain, pain area and the sensory pain response index of the McGill pain questionnaire were all significantly reduced by co-injection of ketamine. The mean PPT values were significantly decreased by approximately 10%, 10, 15 and 25 min after injection of glutamate, but only over the site of injection. Co-injection of ketamine with glutamate also completely blocked the glutamate-induced mechanical sensitization 15 min post-injection as compared with glutamate alone. The lack of spread of mechanical sensitization outside the area of glutamate injection is consistent with the view that glutamate-induced mechanical sensitization results from a peripheral mechanism. The attenuation of glutamate-induced mechanical sensitization by ketamine suggests that this effect is mediated, in part, through activation of peripheral NMDA receptors.