-Lactoglobulin (-LG) is a bovine milk protein sensitive to thermal denaturation. Previously, we demonstrated that such structural change can be detected by a monoclonal antibody (mAb) specific to denatured -LG. In the present study, we show a dramatic increase in -LG immunoreactivity when heating raw milk between 70 and 80°C. To map out the specific epitope of -LG recognized by this mAb, we used a combined strategy including tryptic and CNBr fragments, chemical modifications (acetylation and carboxymethylation), peptide array containing in situ synthesized peptides, and a synthetic soluble peptide for immunoassays. The antigenic determinant we defined was exactly located within the D strand (residues 66 -76) of -LG. Circular dichroic spectral analysis shows that carboxymethylation on -LG not only resulted in a substantial loss of -configuration but also exerted a 10 times increase in immunoreactivity as compared with heated -LG. The result suggests that a further disordered structure occurred in -LG and thus rendered the mAb recognition. Mutations on each charged residue (three Lys and one Glu) revealed that Lys-69 and Glu-74 were extremely essential in maintaining the antigenic structure. We also show an inverse relationship between the immunoreactivity in heated -LG and its binding to retinol or palmitic acid. Most interestingly, pH 9 -10, which neutralizes the Lys groups of -LG, not only reduced its immunoreactivity but also its binding to palmitic acid implicating a role of Lys-69. Taken together, we concluded that strand D of -LG participated in the thermal denaturation between 70 and 80°C and the binding to retinol and palmitic acid. The antigenic and biochemical roles of mAb specific to D strand are discussed in detail.