Association constants for the binding of methyl a ~ galactopyranoside (methyl a-D-Galp) and methyl 2-acetamido-2-deoxy-a-~-galactopyranoside (methyl a-DGalNAcp) to three Bandeiraea simplicifolia isolectins (&, AZB2, B4) were determined by equilibrium dialysis and fluorescence enhancement measurements. The A and B subunits appear to have approximately the same KaesOc for methyl a-D-Galp: 1.45 X lo4, 1.98 X lo4, and 2.06 X lo4 M" for &, A2B2, and B4, respectively, as determined by equilibrium dialysis. Fluorescence enhancement measurements on B4 gave an association constant of 2.07 X lo4 M" for methyl a-D-Galp and 1.87 X LO3 M-' for methyl P-D-Galp. By equilibrium dialysis, we were able to detect 3.3 (theory, 4.0) methyl a-DGaMAcp binding sites for & (Kassoc = 1.87 X lo5 M-'), 1.9 for A2Bz (K,,,,, = 1.19 X lo5 M-'), and were unable to detect any methyl a-D-GalNAcp binding sites for B4. However, four very weak methyl a-D-GalNAc binding sites for B4 were detected by fluorescence enhancement measurement (Kassoc = 1.26 X 10' "I). Thus, the A subunit has an affinity for methyl a-D-GalNAc 3 orders of magnitude greater than the B subunit. Precipitation and hapten inhibition data are in accord with these binding measurements. Toward guaran and type B blood group substance, all isolectins precipitated the same amount of biopolymer. However, AB3, A2B2, and A3B, which are mono-, di-, and trivalent for a-D-GaMAcp, were differentially precipitated by type A blood group substance which contains a-D-GalNAcpend groups. A3B precipitated the most, AzBz less, and AB3 no type A substance. These isolectins should prove useful in studies evaluating the effect of valence on lectin-cell interaction.