[18F]MPPF is a selective serotonin-1A (5-HT1A) receptor antagonist and may be used to measure changes in the functional levels of serotonin (5-HT). The technique is based on the assumption that the injected radiolabeled ligand competes for the same receptor as the endogenous transmitter. Results from studies using serotonergic ligands are not always consistent. The aim of the present study was to investigate if [18F]MPPF binding is decreased after an increase in 5-HT levels. [18F]MPPF binding was assessed in conscious rats using ex vivo autoradiography. We studied the effect of the 5-HT-releasing agent and reuptake inhibitor fenfluramine (10 mg/kg i.p.) and of a combination of the selective serotonin reuptake inhibitor (SSRI) citalopram (10 micromol/kg, s.c.) with the 5-HT2C antagonist ketanserin (100 nmol/kg, s.c). The effect of both treatments on extracellular 5-HT levels was determined using microdialysis. Fenfluramine treatment resulted in a 30-fold increase in extracellular 5-HT levels in the ventral hippocampus and induced a significant reduction of [18F]MPPF binding in the frontal cortex, hypothalamus, amygdala, and hippocampus. The microdialysis results showed a 10-fold 5-HT increase in the ventral hippocampus after combined administration of ketanserin and citalopram. The combination, however, did not affect [18F]MPPF binding. Our data show that [18F]MPPF binding in conscious rats is only reduced after substantial and therefore nonphysiological increases in 5-HT levels. These results may imply that the majority of 5-HT1A receptors is in the low-affinity state, in vivo.