Solutions of beta-lactoglobulin (approximately 1%) at pH 2.5, 4.5, and 6.5 were heated at 90 C for 30 min, cooled rapidly to room temperature (23 to 25 C), and examined for nature and extent of denaturation by various methods. Heating at pH 4.5 and at 6.5 caused almost complete denaturation as judged by loss of solubility at pH of heating, at pH 4.5, or in 2% trichloroacetic acid. However, after heating at pH 2.5 the protein remained in solution but partially lost solubility in 2% trichloroacetic acid and at pH 4.5. The protein fraction denatured by heating at pH 2.5, rendered insoluable at pH 4.5, was unfolded partially and irreversibly, since when resolubilized at pH 2.5 it showed increased optical levorotation and demonstrated no endothermic reaction by differential scanning calorimetry. This material was eluted at lower eluant volumes from a Sephadex G-100 column. However, it was unchanged in molecular size as judged by electrophoretic analysis and measurements of sedimentation velocity. The pH 4.5-soluble fraction from the sample heated at pH 2.5 was similar to native beta-lactoglobulin by the above methods.