The influence of pH and urea concentration on the electrophoretic mobility of native and reduced human serum albumin was evaluated by zonal electrophoresis across transverse urea gradients as well as by migration across transverse pH gradients in gels containing varying concentrations of urea. Exposure to urea results in a change of both pI and hydrodynamic volume of the albumin molecule. At acidic pH, the former effect is brought about by lower urea concentrations than the latter, as made evident by a biphasic denaturation curve; in alkaline buffers, all structural transitions occur at once, and a typical sigmoidal curve is observed. Below pH 6, the lower the pH, the lower the urea concentration causing albumin denaturation. For instance, in the presence of 3 M urea, below pH 5 > 95% of the protein is present in its denatured state, above pH 8 > 90% is in its native form, whereas in the 6.5-7.5 pH range the two components have similar abundance. Also, the reversibility of the transition between folding and unfolding depends upon pH, and is complete only above pH 6. After inclusion of beta-mercaptoethanol in the albumin sample the urea concentration required to bring about protein unfolding increases between pH 4 and 6 and decreases thereafter.