The purpose of this study was to investigate the role of serotonin (5HT) in patients with analgesic-induced headache (AIH). We estimated platelet 5HT concentration in patients with AIH, migraine patients and non- headache controls, by using high performance liquid chromatography with electrochemical detection. Our results revealed a significant decrease (p < 0.00 ) in platelet 5HT content in patients with AIH as compared to migraine patients and non-headache controls (221.8 ± 30.7, 445.3 ± 37.4 and 467.2 ± 38.5 ng/109 platelets, respectively). In contrast, a difference of lesser statistical significance (p=0.022) was observed in platelet 5HT content after incubation with excess 5HT (1940.0 ± 195.1, 2610.0 ± 173.1 and 2560 + 165.2 ng/109 platelets for patients with AIH, migraine patients and non-headache controls, respectively). These data suggest that analgesic-induced suppression of 5HT uptake may interfere with the function of the pain modulatory system in the brainstem. Although the process by which analgesics interfere with this system is as vet unknown, it is possible that it may not be entirely due to defective 5HT uptake mechanisms.