Abstract Background: Central serotonin dysfunction is thought to be involved in the etiology of major depression. Serotonergic challenge studies before and after treatment of depressed patients have yielded conflicting results; however, these studies have not focused on the effect of antidepressant treatment with selective serotonin reuptake inhibitors (SSRIs) on serotonergic challenge studies. Methods: The authors studied 19 outpatients with major depressive disorder using prolactin response to d-fenfluramine as a measure of central serotonergic functioning. Testing of patients was conducted just before and right after 8 weeks of treatment with either fluoxetine ( n = 10) or fluvoxamine ( n = 9) as part of a randomized, double-blind treatment trial. Blood samples for prolactin were collected prior to administration of d-fenfluramine (0.5 mg/kg) and then over the next 5 hours. Results: Unlike previous studies in which antidepressant treatment produced an enhanced prolactin response to fenfluramine, in this study there was no increase in prolactin response to d-fenfluramine following SSRI treatment. In fact, prolactin response to d-fenfluramine was significantly diminished after treatment with fluvoxamine but not fluoxetine. Conclusions: The implications of these findings are discussed with regard to possible mechanisms of action of SSRI treatment.