The effect of subcutaneous injection of glutamate on the mechanical sensitivity of rat facial cutaneous mechanoreceptors was examined. Individual facial mechanoreceptors were recorded in the trigeminal ganglion of anesthetized Sprague-Dawley rats. An electronic von Frey hair was used to measure the mechanical threshold (MT) of the afferent fibers at baseline and following subcutaneous injection of glutamate (0, 0.01, 0.1, 1M; 10microl) or glutamate (0, 0.1M) plus the competitive N-methyl-d-aspartate (NMDA) receptor antagonist 2-amino-5-phosphonovalerate (APV; 0.01M). Subcutaneous injections were randomized and the investigator was unaware of their content. Changes in MT were assessed with a repeated measure ANOVA with time, sex and treatment as factors. Immunohistochemistry was used to confirm NMDA receptor expression by cutaneous nerve fibers. A total of 100 (50 per sex) facial mechanoreceptors were recorded from 61 (32 females, 29 males) rats in two separate experiments. Subcutaneous injections of higher concentrations of glutamate (1, 0.1M) induced a significant mechanical sensitization of skin afferent fibers (compared to 0 and 0.01M). Females (EC(50)=16.2mM) were more sensitive to glutamate than males (EC(50)=73.0mM). Facial cutaneous nerve fibers in both sexes expressed NMDA receptors. APV blocked the mechanical sensitization of the afferent fibers treated by glutamate 0.1M in both sexes with a lower effect in females at a 10-20minute post-injection. Subcutaneous injection of glutamate mechanically sensitizes rat facial cutaneous mechanoreceptors through activation of peripheral NMDA receptors. Peripheral NMDA receptor antagonists may be considered for craniofacial pain.