A new three-phase separation phenomenon was observed, in which two water-immiscible liquid phases (a first and a second phase) were immediately formed from a homogeneous aqueous solution, This phenomenon appeared on the charge neutralization (e.g., pK(a) value of HPFOA was 1.01, at I = 0.1, 20 degrees C) of the perfluorooctanoate ion (PFOA(-)) with a proton (H+) in aqueous solution containing a small amount of a water-miscible organic solvent such as dimethyl sulfoxide (DMSO) and PFOA(-) ion. The optimum conditions for the three-phase separation behavior were defined as follows : the pH was 0.42 to 0.46, the total concentration of PFOA(-) ion was 0.025 to 0.032 mol dm(-3), and that of DMSO was 0.90 to 2.43 mol dm(-3).