Hydrophobic binding to the lectin from lima beans (Phaseolus lunatus) was studied by lectin-induced alterations in the fluorescence and absorption spectra of several hydrophobic ligands. The fluorescence of 1,8-anilinonaphthalenesulfonic acid (ANS) was greatly enhanced in the presence of lima bean lectin with concomitant shift of the fluorescence emission maximum from 520 to 469 nm. Similar enhancement was seen with 2,6-toluidinylnaphthalenesulfonic acid (TNS) with a shift of emission from 500 to 423 nm, and with an uncharged analogue, N-phenyl-1-naphthylamine. Fluorescence titrations with ANS and rose bengal yielded affinity constants of 3.9 X 10(3) and 6 X 10(5) M-1, respectively. Fluorescence titration with TNS indicated binding heterogeneity and yielded intrinsic association constants of 7.8 X 10(4) and 2.2 X 10(3) M-1 assuming a model with two classes of independent sites. The high affinity binding had an apparent stoichiometry of 1.08 sites/lectin tetramer. Equilibrium dialysis for ANS and TNS confirmed the results of fluorescence titration and gave stoichiometries for ANS and low affinity TNS binding of one site/subunit. Neither chemical modification of thiol groups known to be essential for carbohydrate binding nor titration in the presence of haptenic sugar affected the binding of ANS or TNS to the lectin. These results indicated that the carbohydrate and hydrophobic binding sites of lima bean lectin are independent and noninteracting.