Aim: To determine if glutamate injected into the healthy temporomandibular joint (TMJ) evokes pain through peripheral N-methyl-D-aspartate (NMDA) receptors and if such pain is influenced by sex or sex steroid hormones. Methods: Sixteen healthy men and 36 healthy women were included and subjected to two randomized and double-blind intra-articular injections of the TMJ. Experimental TMJ pain was induced by injection of glutamate (1.0 mol/L) and NMDA block was achieved by co-injection of the NMDA antagonist ketamine (10 mmol/L). The TMJ pain intensity in the joint before and during a 25-minute postinjection period was continuously recorded on an electronic visual analog scale (0 to 10). Estradiol, progesterone, and testosterone levels in serum were analyzed. Results: Glutamate-induced pain showed a median (25/75 percentile) duration of 8.3 (5.2/12.2) minutes. The peak pain intensity was 6.1 (4.2/8.2), the time to peak was 50 (30/95) seconds, and the area under the curve was 59 (29/115) arbitrary units. The women reported higher maximum pain intensity than the men and shorter time to peak. The sex hormone levels were not significantly related to the glutamate-induced TMJ pain. NMDA block significantly reduced the glutamate-induced TMJ pain, mainly in the women. There were no significant correlations between sex hormone levels and the effects of NMDA block for any pain variable. Conclusion: Glutamate evokes immediate pain in the healthy human TMJ that is partly mediated by peripheral NMDA receptors in the TMJ. J Orofac Pain 2010;24:172–180