Serotonin (5-hydroxytryptamine, 5-HT) is known to play a role in a wide variety of functions including: mood, anxiety, sleep, aggression, sexual function and cognitive function. In the past decade, the sudden interest in acute amino acid depletion paradigms has stimulated the interest in studying the function of 5-HT in man in vivo. In particular, investigating the association of experimentally lowered 5-HT with cognition by measuring the influence of acute tryptophan depletion (ATD) on performance, and in some cases applying ATD as a challenge test using cognition as disease surrogate marker, have recently become popular. The crucial questions, ‘what can this method tell us about cognitive function in health and disease?’ and ‘what can ATD-induced cognitive changes tell us about health and disease?’ can only be answered after first considering basic assumptions about the method. These pertain to the derived questions, ‘is ATD a serotonergic manipulation?’, ‘is the placebo used in ATD an inactive treatment?’. Also, ‘are there specific cognitive markers of ATD?’. After a brief consideration of these issues, we will focus on the more clinical issues addressing the question whether ATD can be used as a challenge test, to indicate serotonergic vulnerability, using cognitive functions as surrogate measures of disease in particular.