Serotonin (5-HT) neurotransmission is involved in the normal regulation of affect and cognition, and its dysfunction is implicated in a range of mental disorders. However, there is currently no method to directly assess 5-HT synaptic levels in the living human brain. In the human dopamine (DA) system, baseline receptor occupancy by endogenous DA can be estimated by measuring the effects of DA depletion on the binding of certain radioligands through endogenous competition, particularly in striatum. For example, the measured increase in 11C-raclopride and 123I-IBZM binding following acute DA depletion implies a baseline D2 receptor occupancy of 10% in healthy humans. Brain 5-HT can be transiently and safely reduced by rapid tryptophan depletion (RTD), a technique involving the ingestion of an amino acid mixture lacking L-tryptophan, the dietary precursor of 5-HT. We have used RTD in healthy humans in an attempt to demonstrate a measurable effect of reduced 5-HT on the specific binding of the selective 5-HT transporter (SERT) ligand 11C-DASB and the selective 5-HT2A receptor antagonist ligand 11C-MDL100907. However, values of regional specific binding were not significantly different between the reduced endogenous 5-HT and control conditions for either tracer. These results suggest that PET with 11C-DASB or 11C-MDL100907 is not sensitive to the decreased concentration of synaptic 5-HT following RTD, and that neither tracer has the potential for measuring changes in synaptic levels of 5-HT in vivo. Imaging techniques relying on endogenous competition would ideally require a brain region with high synaptic levels of, and significant baseline receptor occupancy by, endogenous neurotransmitter. However, in vivo microdialysis studies in non-human primates suggest that extracellular 5-HT concentration is very low (< 0.5 nM) across brain regions and is 1 – 2 orders of magnitude lower than striatal DA levels. In the case of the 5-HT2A receptor, this concentration is significantly below the estimated KD of 5-HT for the receptor configured in a state of high affinity for agonists ( 1.3 nM). Moreover, in vitro studies suggest that the proportion of receptors configured in a state of high affinity for agonists, and therefore potentially available to endogenous 5-HT, is low ( 13%). Together, these values suggest that baseline occupancy of 5-HT2A receptors by 5-HT may be only approximately 2%, making changes following increased or decreased 5-HT unmeasurable by current techniques in humans. Agonist serotonergic radioligands may be expected to be superior for endogenous competition studies, but have so far been unsuccessful in humans. Efforts to experimentally increase baseline receptor occupancy (for example by pretreatment with selective 5-HT reuptake inhibitor medication) prior to RTD may be of interest. However, the insensitivity of current tracers to endogenous competition implies that they may be used to measure differences in SERT or 5-HT2A receptor availability between populations or during therapy without the need to consider concomitant changes in neurotransmitter concentration.