The objective of the study was to investigate whether addition of acidifier (A) to a diet containing crystalline tryptophan (TRP) does not impair utilization of the amino acid both for growth and serotonin synthesis in broiler chicken. Eight groups, each of 12 eight-day-old, individually housed, female broilers, were fed on diet containing 1.57 g TRP/kg, or diets supplemented with 0.21, 0.42 and 0.63 g TRP, without or with 3 g acidifier (A - mixture of phosphoric, citric and fumaric acids) per kg. Feed intake and body weight (BW) was registered for 3 weeks, followed by balance experiment, at the end the birds were killed and digesta and brain tissue were sampled. TRP supplementation increased BW gain and improved feed utilization (P<0.05), and did not affect ileal, while slightly decreased faecal protein digestibility. The concentration of serotonin (5-HT) and the ratio of 5-hydroxy-indole-3-acetic acid (5-HIAA) to 5-HT in brain tissue increased (P<0.01) with the increase of TRP level in diet. Supplementation with A had no significant effect on performance, pH of digesta, ileal and faecal protein digestibility in comparison with unsupplemented diets. However, it lowered the concentration of 5-HT (2.75 vs 2.98 μg/g; P<0.05) and increased the ratio of 5-HIAA/5-HT (0.30 vs 0.25; P<0.01) in brain tissue, the negative effect was greatest in TRP deficient diet (TRP × A; P<0.001). It indicate for a need of careful adjusting TRP level in broiler diets supplemented with acidifiers.