The aim of this study was to determine if peptides could interact with beta-lactoglobulin (beta-LG) and what the physicochemical conditions promoting their interaction with the protein are. The binding of negatively charged (beta-LG 125-135 and 130-135), positively charged (beta-LG 69-83 and 146-149), and hydrophobic (alphaS1-CN 23-34 and beta-LG 102-105, both bioactive peptides) peptides to bovine beta-LG was determined using an ultrafiltration method under different physicochemical conditions: pH 3.0, 6.8, and 8.0; buffers of 0.05 and 0.1 M; 4, 25, and 40 degrees C; beta-LG/peptide ratios of 1:5 and 1:10. At pH 3.0, none of the peptides interacted with beta-LG at any temperature, buffer molarity, or beta-LG/peptide ratio probably due to electrostatic repulsions between the highly protonated species. At pH 6.8 and 8.0, charged peptides beta-LG 130-135, 69-83, and 146-149 bound to beta-LG under some physicochemical conditions, possibly by nonspecific binding. However, both hydrophobic peptides probably bind to the inner cavity (beta-barrel) of beta-LG, provoking the release of materials absorbing at 214 nm. Given the known biological activities of the hydrophobic peptides used in this study (opioid and ACE-inhibitory activities), their binding to beta-LG may be relevant to a better understanding of the physiological function of the protein.