The equilibrium binding of the apolar fluorescent dye 1-anilinonaphthalene-8-sulphonate (ANS) to bacteriorhodopsin, BSA, chicken egg lysozyme, ovalbumin, porcine somatotrophin (PST) and bovine pancreatic ribonuclease (RNAase) was quantitatively evaluated using Scatchard- and Klotz-plot analyses. On the basis of the average association constant for ANS binding sites (Ka), the proteins could be ranked in order of surface hydrophobicity as: Bacteriorhodopsin greater than BSA greater than ovalbumin greater than PST greater than lysozyme greater than RNAase. The number of protein-ANS binding sites was determined as 54, 10, 3, 1, 2 and 1 respectively. The ANS-based assessment of the surface hydrophobicity of these proteins was generally in agreement with the average hydrophobicity based on amino acid sequence [Bigelow (1967) J. Theor. Biol. 16, 187-211], except for results with PST and ovalbumin. The proteins were also analysed by reversed-phase h.p.l.c. using C1 and C8 columns. There was no significant correlation between ANS and reversed-phase-h.p.l.c. assessment of hydrophobicity, with the results obtained by h.p.l.c. being dependent upon the column used. ANS-based measurement of surface hydrophobicity appears to be the most appropriate means for assessing proteins such as to reflect their overall three-dimensional structure in solution.