For investigation of the conformation of the unfolded species and its role in the refolding kinetics, refolding kinetic measurements were made on hen egg-white lysozyme by using the stopped-flow method at 25 degrees C in the four sets of initial and final folding condition: (1) 4 M guanidinium chloride (GdmCl) and 0.5 M GdmCl; (2) 40% acetic acid (HOAc) and 5% HOAc; (3) 4 M GdmCl and 0.5 M GdmCl-5% HOAc; (4) 40% HOAc and 0.5 M GdmCl-5% HOAc. The kinetic results as measured by absorbance at three wavelengths, 301, 292, 250 nm, agreed with each other and indicated strict biphasic behavior without exception. The kinetic parameters were determined only by the final refolding conditions. The spectral properties of the unfolded species at the end of stopped-flow mixing were investigated by comparing the total kinetic amplitude with the difference between the static absorbance of the native molecule in the final refolding conditions and that of the unfolded molecule in the initial unfolding conditions. The solvent effect was considered in the comparison. It was concluded that the unfolded species assumed a new transient conformation in the mixing process and that the transformation was completed within the mixing time.