beta-Lactoglobulin (beta-LG) is a lipocalin, which is the major whey protein of cow's milk and the milk of other mammals. However, it is absent from human milk. The biological function of beta-LG is not clear, but its potential role in carrying fatty acids through the digestive tract has been suggested. beta-LG has been found in complexes with lipids such as butyric and oleic acids and has a high affinity for a wide variety of compounds. Serotonin (5-hydroxytryptamine, 5-HT), an important compound found in animals and plants, has various functions, including the regulation of mood, appetite, sleep, muscle contraction, and some cognitive functions such as memory and learning. In this study, the interaction of serotonin and one of its derivatives, arachidonyl serotonin (AA-5HT), with beta-LG was investigated using circular dichroism (CD) and fluorescence intensity measurements. These two ligands interact with beta-LG forming equimolar complexes. The binding constant for the serotonin/beta-LG interaction is between 10(5) and 10(6) M(-1), whereas for the AA-5HT/beta-LG complex it is between 10(4) and 10(5) M(-1) as determined by measurements of either protein or ligand fluorescence. The observed binding affinities were higher in hydroethanolic media (25% EtOH). The interactions between serotonin/beta-LG and AA-5HT/beta-LG may compete with self-association (micellization) of both the ligand and the protein. According to far-and near-UVCD results, these ligands have no apparent influence on beta-LG secondary structure, however they partially destabilize its tertiary structure. Their binding by beta-LG may be one of the peripheral mechanisms of the regulation of the content of serotonin and its derivatives in the bowel of milk-fed animals.