Aims: To document and compare the intensity, localization, and quality of pain evoked by glutamate injections into the human masseter or splenius muscles and to determine the effect of glutamate-evoked pain on the pressure pain thresholds (PPTs) in both jaw and neck muscles. Methods: Twenty-six healthy men were given painful injections of glutamate (1.0 mol/L) and control injections of isotonic saline (0.165 mol/L) into the masseter and splenius muscles. The subjects rated the perceived intensity of pain on a visual analog scale (VAS), drew the area of the pain on maps of the face and neck, and filled out a Danish version of the McGill Pain Questionnaire (MPQ). PPTs were used to assess the sensitivity of the masseter and splenius muscles to mechanical stimuli (n = 11). Results: Glutamate injection into the masseter or splenius evoked pain lasting almost 10 minutes. Peak pain intensity usually occurred within 2 minutes of the injection, and VAS scores of peak pain were significantly higher for the masseter muscle compared with the splenius muscle (paired t test, P = .003). The pain area from the masseter injections did not extend into the neck region, although in some subjects the pain from the neck region extended into the temporal region. There were no significant relationships between the area of perceived pain and the VAS pain scores (Pearson correlation, P > .297). Glutamate-evoked pain in either the masseter or splenius muscles was associated with significant decreases in masseter or splenius PPTs, respectively (2-wayANOVAs, P .016). Isometric saline injections were almost pain-free and caused no PPT changes. Conclusion: The data suggest that the masseter muscle is more sensitive to glutamate injections and mechanical stimuli than the splenius muscle. The relatively limited overlap between the sensory manifestations of pain from masseter and splenius muscles may have potential implications for diagnosis and management of myofascial pain complaints in the craniofacial and neck region. Keywords: cervical spine pain disorders, experimental muscle pain, glutamate, temporomandibular disorders, trigeminal pain