Pain in myofascial temporomandibular disorder (TMD) can affect both the masseter and temporalis muscles. Glutamate injection into the masseter muscle evokes pain that is greater in men than in women and this pain is attenuated by co-injection of the N-methyl-d-aspartate (NMDA) receptor antagonist ketamine (10 mmol/L) in men. Animal studies suggested that pain induced by peripheral NMDA receptor activation could differ between the temporalis and masseter muscles and between men and women. The study aims were to investigate differences in glutamate-evoked pain between these muscles and the effectiveness of ketamine to attenuate glutamate-evoked pain in both genders. Pain and mechanical sensitivity were induced in 2 sessions of an experiment in 14 women and 16 men by repeated injections of glutamate (0.5 mol/L) with and without ketamine (20 mmol/L) into the masseter and temporalis muscles. Two injections were applied into the same masseter muscle and 2 injections into the same anterior temporalis muscle at each session. Visual analogue scale (VAS) pain intensities and pain drawing areas were assessed. Glutamate-evoked pain and pain drawing area were significantly greater from the temporalis muscle than from the masseter muscle (P <.02) in both genders. Women reported significantly greater glutamate-evoked masseter muscle pain than men (P <.03). Co-injection of ketamine, at higher dose than previously used, was equally effective in attenuating glutamate-evoked pain from both muscles in both genders (P <.01). The current findings indicate that the characteristics of pain generated by intramuscular injection of glutamate vary for different masticatory muscles and may be partially generated through activation of peripheral NMDA receptors. © 2011 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.