The method of acute tryptophan depletion (ATD), which reduces the availability of the essential amino acid tryptophan (TRP), the dietary serotonin (5-hydroxytryptamine (5-HT)) precursor, has been applied in many experimental studies. ATD application leads to decreased availability of TRP in the brain and its synthesis into 5-HT. It is therefore assumed that a decrease in 5-HT release and subsequent blunted neurotransmission is the underlying mechanism for the behavioural effects of ATD. However, direct evidence that ATD decreases extracellular 5-HT concentrations is lacking. Furthermore, several studies provide support for alternative underlying mechanisms of ATD. This may question the utility of the method as a selective serotonergic challenge tool. As ATD is extensively used for investigating the role of 5-HT in cognitive functions and psychiatric disorders, the potential of alternative mechanisms and possible confounding factors should be taken into account. It is suggested that caution is required when interpreting ATD effects in terms of a selective serotonergic effect.