Trifluoroacetic acid (0.1% w/v) is an excellent solvent for polypeptides, is volatile, and has a low absorbance of ultraviolet light of low wavelength. Polypeptides subjected to chromatography on columns of TSK-G2000-SW or TSK-G3000-SW in this solvent were eluted as sharp peaks. Retention volume was dependent not only on molecular weight but also on the number of formal charges per molecule. For polypeptides with a similar molecular weight, that with the highest proportion of basic amino acid residues was eluted earliest. For the TSK-G2000-SW column, the degree of deviation from a linear relationship between elution volume and logarithm of molecular weight was directly proportional to the molecular weight to the power 2/3 divided by the number of positive charges per molecule. Inclusion of 0.25 M sodium chloride in the solvent increased both the upper and lower limits of the molecular weight range over which separation occurred. The use of 0.1% trifluoroacetic acid as the mobile phase is thus particularly applicable to the separation of peptides of low molecular weight.