Serotonin (5-HT, 5-hydroxytryptamine) may have an important role in the maintenance of normal neuropsychological functioning. The method of acute tryptophan depletion (ATD) provides a pharmacological challenge by which central 5-HT levels can be temporarily decreased and effects on learning, memory and mood examined. Twenty healthy male volunteers were recruited to take part in this within-subject, double-blind, crossover study. Neuropsychological function was evaluated 4-6 h after ingestion of a control or 52 g tryptophan (TRP) depleting amino-acid drink. ATD significantly lowered levels of plasma total and free TRP (p < 0.001), but this did not affect mood or performance on tests of verbal and visuo-spatial learning and memory, attention or executive function. These results contradict previous findings; however, the degree of disruption of central 5-HT levels resulting from the use of the 52 g amino-acid protocol may be an important factor in explaining the lack of effect. By utilizing more specific probes of individual 5-HT receptor subtypes, future studies can fully explore the role of 5-HT in neuropsychological functioning and may elucidate the factors determining vulnerability to the effects of serotonergic dysfunction.