Aims: Serum serotonin levels (S-5-HT) have been reported to be reduced in patients with fibromyalgia and to show a negative correlation with pain. We hypothesized that one mechanism behind this could be that platelets are activated to release 5-HT into the plasma compartment (P-5-HT), which then binds to nociceptors. The aims of this study were therefore to investigate the relation between P-5-HT and S-5-HT and their relationship versus orofacial pain and anxiety in fibromyalgia. Methods: Twelve patients with fibromyalgia, 12 patients with rheumatoid arthritis, and 12 healthy individuals participated in the study. Pain measures used were pain intensity assessed with a visual analog scale, pain drawings, and influence of pain on daily living activities (ADL). The Spielberger State and Trait Anxiety Inventory (STAI) scale was used for self-rating of anxiety levels. The participants were examined clinically, and the pressure pain threshold (PPT) over the masseter muscle was assessed. Finally, venous blood was collected for analysis of P-5-HT and S-5-HT. Results: The ratio between P-5-HT and S-5-HT was calculated to determine the relative plasma fraction of serotonin (RPS). Patients with fibromyalgia showed significantly lower S-5-HT than did patients with rheumatoid arthritis. They also showed significantly higher STAI scores and tender point index of orofacial muscles and significantly lower PPT than the healthy individuals. High RPS was associated with high ADL and STAI scores. Conclusion: This study indicates that a high level of plasma serotonin in relation to serum level is associated with pain discomfort and increased anxiety in fibromyalgia.