Identification of a biomarker that can inform on extracellular serotonin (5-HT) levels in the brains of living humans would enable greater understanding of the way brain circuits are modulated by serotonergic neurotransmission. Substantial evidence from studies in animals and humans indicates an inverse relationship between central 5-HT tonus and 5-HT type 4 receptor (5-HT 4 R) density, suggesting that 5-HT 4 R receptor density may be a biomarker marker for 5-HT tonus. Here, we investigated whether a 3-week administration of a selective serotonin reuptake inhibitor, expected to increase brain 5-HT levels, is associated with a decline in brain 5-HT 4 R binding. A total of 35 healthy men were studied in a placebo-controlled, randomized, double-blind study. Participants were assigned to receive 3 weeks of oral dosing with placebo or fluoxetine, 40 mg per day. Brain 5-HT 4 R binding was quantified at baseline and at follow-up with [11 C] SB207145 positron emission tomography (PET). Three weeks of intervention with fluoxetine was associated with a 5.2% reduction in brain 5-HT 4 R binding (P= 0.017), whereas placebo intervention did not change 5-HT 4 R binding (P= 0.52). Our findings are consistent with a model, wherein the 5-HT 4 R density adjusts to changes in the extracellular 5-HT tonus. Our data demonstrate for the first time in humans that the imaging of central 5-HT 4 R binding may be used as an in vivo biomarker of the central 5-HT tonus.