The pathophysiology behind chronic pain from masticatory muscles is unclear. Our hypothesis was that this pain is of inflammatory origin and associated with release of inflammatory mediators. The aim of this study was therefore to investigate the presence of prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) in the masseter muscle and plasma and their relation to myalgia. Nineteen patients with fibromyalgia, 19 with local myalgia of the masseter muscle, and 11 healthy individuals were examined with regard to local muscular pain intensity at rest and pressure pain threshold. Inclusion criteria were masseter muscle pain for at least 3 months and masseter muscle tenderness on digital palpation. Samples were obtained from the masseter muscle by microdialysis, and the dialysates and venous blood samples were analyzed with regard to PGE2 and LTB4 concentration. Intramuscular levels were found in all groups, with significantly higher levels of LTB4 in the patients with fibromyalgia, in whom PGE2 was positively correlated to muscular pain. In the healthy individuals PGE2 was negatively correlated to pressure pain threshold. In both patient groups but not in the healthy individuals LTB4 increased during the consecutive samplings. PGE2 and LTB4 were detectable in the plasma of all groups. In conclusion, both PGE2 and LTB4 were found in the human masseter muscle. LTB4 levels are increased on needle trauma in patients with myalgia. PGE2 levels are related to muscular pain in patients with fibromyalgia. Masseter muscle pain therefore seems to be partly of peripheral inflammatory origin in fibromyalgia.