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According to the monoamine hypothesis, the development of depression is associated with dysfunctions of the serotonergic system. Alterations in the serotonin transporter gene (5-HTTLPR), the serotonergic activity in the brain, and the content of serotonin (5-HT) have been related to depression and were examined separately by previous studies. This study investigates these parameters in 89 depressed patients and 89 healthy participants. We investigated the serotonergic activity measured by the loudness dependence of auditory evoked potentials (LDAEP). In addition to the examination of the serotonin content (serum and platelet), enzyme-linked immunosorbent assays (ELISA) were used and 5-HTTLPR genotypes were analyzed. We observed a lower serotonin content in patients compared to healthy participants. Further, we noticed a correlation between anxiety and depression-associated symptoms with serotonergic activity. Patients treated with SSRI/SNRI showed decreased contents of serum serotonin compared to patients without any psychotropic medication or other psychotropic medications. Since the serotonergic activity, peripheral serotonin content, and 5-HTTLPR were unrelated, the results suggest independent alterations of central and peripheral serotonergic systems in depression. In line with this finding, serotonergic activity was related to anxiety and depression symptoms. Furthermore, the applied medication seems to influence serum serotonin content in patients with depression.