A mixture of amino acids (arginine, phenylalanine, alanine, glycine and aspartic acid) in solution was extracted by four acidic extractants (dinonylnaphthalenesulphonic acid (DNNSA), di(2-ethylhexyl)monothiophosphoric acid (D2EHPA(S)), di(2-ethylhexyl)phosphoric acid (D2EHPA) and Versatic 10) in toluene. The extractive capacity of the organic phase for the amino acids, using DNNSA, D2EHPA(S) and D2EHPA was found to decrease in the order arginine > phenylalanine > alanine > glycine > aspartic acid, although at low pH values phenylalanine > arginine occurred for D2EHPA(S) and D2EHPA. Separation factors derived for pairs of amino acids were in the range 2.0 (glycine-aspartic acid) to 20.1 (alanine-glycine). The extractive and loading capability of the extractants for the amino acids was found to decrease in the order DNNSA > D2EHPA(S) > D2EHPA > Versatic 10, which follows the reverse order of their respective acid dissociation constants. DNNSA was shown to be a promising extractant for the extraction and fractionation of amino acids.