Serotonin (5-HT) was implicated in both clinical and experimental studies in flexible, goal-directed behavior. However, the way in which 5-HT manipulations affect brain activation patterns underlying different subprocesses of cognitive flexibility remains largely unknown. OBJECTIVES: The aim of this study was to investigate the effect of a transient lowering of 5-HT on brain activation during performance monitoring and response inhibition. MATERIALS AND METHODS: We used acute tryptophan depletion (ATD), a well-known method to reduce central 5-HT, to investigate the effect of a transient lowering of 5-HT on the blood-oxygen-level dependent (BOLD) response in an event-related functional MRI study. Thirteen healthy male volunteers performed a modified Go/NoGo task in a counterbalanced, placebo-controlled, within-subject design. RESULTS: ATD significantly lowered plasma tryptophan but did not affect mood and cognitive performance. ATD decreased the BOLD response in the dorsomedial prefrontal cortex (BA 8) during performance monitoring. ATD did not affect the BOLD response during response inhibition. CONCLUSIONS: This study provides more evidence for the suggested role of 5-HT in performance monitoring. Because ATD studies have revealed inconsistent effects of ATD on performance and on brain activation, it was suggested that gender and personality traits are important variables to take into account for future research.