Polymyalgia rheumatica (PMR) is characterized by aching proximal muscles and systemic inflammation. We explored the pain-eliciting mechanisms by measuring interstitial levels in muscle of potentially pain-inducing substances as well as local blood flow. Twenty glucocorticoid-naive patients with newly diagnosed PMR and 20 controls were examined before and after 14 days of prednisolone (20 mg/day). Concentrations of glutamate, prostaglandin E2 (PGE2), bradykinin, serotonin, adenosine triphosphate, lactate, pyruvate, and potassium as well as extraction of 3H2O were measured in symptomatic vastus lateralis and trapezius muscles using microdialysis. Plasma levels were measured simultaneously. To be considered potentially pain inducing, interstitial concentrations of candidates should be higher in patients vs. controls, be normalized by prednisolone, and be higher in muscle vs. plasma. Prednisolone abolished symptoms in all patients within 2 days. Before treatment glutamate in both muscles (vastus: 60 ± 7 vs. 38 ± 7 µmol/L; trapezius: 60 ± 6 vs. 43 ± 7 µmol/L) and PGE2 in vastus (911 ± 200 vs. 496 ± 122 pg/mL) were higher in patients than in controls (P < 0.05), and higher in muscle than in plasma (P < 0.05). Prednisolone abolished the differences between patients and controls. No other candidate completely fulfilled the predefined requirements for pain-inducing substances in PMR. 3H2O extraction was identical between groups. In conclusion, local release of glutamate and PGE2, but not ischemia, may contribute to the muscle pain in PMR. This supports the view that intramuscular mechanisms are important in PMR.