The essential amino acid tryptophan is the precursor of the neurotransmitter serotonin. By depleting the body of tryptophan, brain tryptophan and serotonin levels are temporarily reduced. In this paper, several experiments are described in which dose and treatment effects of acute tryptophan depletion (ATD) using a gelatin-based protein-carbohydrate mixture were studied in male and female Wistar rats. Two or three doses of tryptophan depleting mixture resulted in 65-70% depletion after 2-4 h in males. ATD effects were similar in females, although females may return to baseline levels faster. Treatment effects after four consecutive days of ATD were similar to the effects of 1 day of treatment. Object recognition memory was impaired 2, 4, and 6 h after the first of two doses of ATD, suggesting that the central effects occurred rapidly and continued at least 6 h, in spite of decreasing treatment effects on plasma tryptophan levels at that time point. The method of acute tryptophan depletion described here can be used to study the relationship between serotonin and behaviour in both male and female rats.