Melatonin is a major biosynthetic product of pineal gland exerting a potent antioxidant and the reactive oxygen metabolites scavenging activities but the mechanism of formation of this indole at extrapineal sources has not been fully elucidated. It is known that the gastrointestinal (GI)-tract plays an important role as a source of melatonin synthesis but the conversion of L-tryptophan into melatonin in the GI-tract of experimental animals and humans should be further examined. In this study, the conversion of L-tryptophan to melatonin was determined in the serum collected from rats administered intragastrically with this amino acid acting as melatonin precursor. For this purpose, a simple, sensitive and reliable method was developed for simultaneous determination of six L-tryptophan metabolites in rat serum, namely, 5-hydroxytryptamnie (5-HT), 5-hydroksytryptophan (5-HTR), kynurenin (KYN), antranilic acid (AA), indole-3-acetic acid (IAA) and melatonin that were analyzed in one chromatographic run by high-performance liquid chromatography (HPLC) with UV and native fluorimetric detection with multiple wavelengths. We used nucleosil Supelco C18 5 μm 4.6 mm x 250 nm column with the standard mobile phase consisting of solvent A (water/0.1% trifluoroacetic acid (TFA) and solvent B (methanol/0.1% TFA) in gradient elution. Fifty five rats received vehicle (saline) of L-tryptophan (50 mg/kg) or melatonin (50 mg/kg) by means of intragastric gavage and they were anesthetized and sacrificed at 0, 10, 20, 30, 60, 120 or 240 min upon L-tryptophan or melatonin administration for the venous blood withdrawal. The serum collected samples were kept on ice for the HPLC determination. The average recovery of 5-HT, 5-HRT, KYN, AA, TRP, IAA, and melatonin were 99±3%, 97±1.5%, 94±2.5%, 99±2.46, 98±1.5 and 98±2%, respectively. We conclude that 1) L-tryptophan is converted to melatonin in the GI-tract during the day when the pineal gland synthesis is inhibited, and 2) the reverse phase high performance liquid chromatography (RP-HPLC) is a new sensitive and reliable method that could be successfully applied to the study of kinetics and metabolism of L-tryptophan in GI-tract.