The relationship between peripheral serotonergic variables, melancholic traits, and clinical improvement after antidepressant treatment was examined in 83 drug-free major depressive patients. Plasma serotonin (5-HT) concentrations was lower in untreated melancholic patients (1.00 ± 0.11 vs. 1.84 ± 0.28 ng/mL, p < 0.008; N = 40 and 43, respectively). A tendency was observed for plasma 5-hydroxyindoleacetic acid (p < 0.06), whereas platelet 5-HT and plasma tryptophan did not differ between groups. After blood sampling and clinical ratings, treatment began with fixed doses of 5-HT uptake inhibitors (clomipramine or fluvoxamine), monoamine oxidase inhibitors, or tianeptine, a 5-HT uptake enhancer. There was no significant difference in response rates between patients with and without melancholic traits. The relationship between the clinical response at 6 weeks (>50% reduction of baseline Hamilton score) and the pretreatment values of biochemical variables was examined. Responders had a lower pretreatment platelet 5-HT (530 ± 36 vs. 664 ± 50 ng/109 platelets, p < 0.03; N = 44 and 39, respectively). Patients with a platelet 5-HT concentration above 800 ng/109 platelets had a lower response rate than those below this value (p < 0.003). This difference was maximal in the subgroup of patients treated with 5-HT uptake inhibitors (N = 49). In this subgroup, the response rates of patients with 5-HT concentrations below and above the cutoff point were, respectively, 70% and 17% (p < 0.001). A pretreatment platelet 5-HT value above 800 ng/109 platelets had a predictive value for a negative response of 92%. These results suggest the presence of biochemical differences in the peripheral serotonergic system between melancholic and non-melancholic patients. The inverse relationship between the pretreatment platelet 5-HT content and clinical response may be useful in the investigation of the relationship between the 5-HT system and antidepressant response.