A new technique, temperature gradient gel electrophoresis (TGGE), was applied to the study of heat-induced protein denaturation. The gels used contained 30 mM Borax + 75 mM boric acid, pH 8.4, and various concentrations of urea. When this technique was applied to bovine serum albumin, the protein showed discontinuous bands upon melting, indicating that the thermal transition is irreversible. The apparent melting temperature, calculated based on the relative intensity of two bands in the transition region, was 58 degrees C in 2 M urea. When the thermal denaturation of bovine serum albumin was analyzed spectroscopically, the transition was again irreversible, with an apparent transition temperature of 57 degrees C, consistent with the TGGE results. Recombinant stem cell factor, recombinant granulocyte macrophage-colony stimulating factor, and catalase were also analyzed by TGGE, indicating that the technique can be used to analyze denaturation of monomeric and multimeric proteins.