The role of the free sulfhydryl group of P-lactoglobulin in the formation of a stable non-native monomer during heat-treatment of beta-lactoglobulin solutions was investigated. Two concomitant events occurred at the earlier stage of heating: unfolding of native globular monomer and intramolecular sulfhydryl/disulfide exchange reaction. Thus, two denatured monomeric species were formed: a non-native monomer with exposed Cys-121 (Mcys121) which became reversible after cooling, and a stable non-native monomer with exposed Cys-119 (Mcys 119) which exhibited both a larger hydrodynamic conformation than native monomer and low solubility at pH 4.7. The results also show that the formation of these monomeric species throughout heat-induced denaturation of native beta-1g monomers is faster than their subsequent aggregation. A mechanism describing the behavior of beta-1g denaturation/aggregation during heat-treatment under selected conditions (5.8 mg/ml, low ionic strength, pH 6.6, 85 degreesC is presented. (C) 2003 Elsevier Science (USA). All rights reserved.