The conformations of the NeuAc alpha 2(I)-->3Gal beta 1(II)-->4[Fuc alpha 1(III)-->3]GlcNAc-O-CH3 tetrasaccharide (sLe(x)), in aqueous solution and bound to E-, P-, and L-selectin have been determined using high resolution NMR spectroscopy. In the free ligand, the conformation of glycosidic linkage I is disordered with {Phi(I), Psi(I)} sampling values close to {-60 degrees, 0 degrees}, {-100 degrees, -50 degrees}, and {180 degrees, 0 degrees}. The trisaccharide portion is rigid and characterized by {Phi(II), Psi(II); Phi(III), Psi(III)} = {46 degrees, 18 degrees; 48 degrees, 24 degrees}. The measured dissociation rates and equilibrium binding constants, {k(off), K-D}, were {164 +/- 24 s(-1), 0.72 +/- 0.4 mM}, {522 +/- 166 s(-1), 7.8 +/- 1.0 mM}, and {1080 +/- 167 s(-1), 3.9 +/- 0.6 mM} at 300 K for E-, P-, and L-selectin, respectively. The bound conformations of the ligand were calculated from the full relaxation matrix analysis of transferred-NOE spectra for E- and P-selectin or by using a two-spin approximation for the L-selectin complex. Both E- and P-selectin recognize the {-60 degrees, 0 degrees} conformation of sLe(x) while the {-100 degrees, -50 degrees} conformer is probably recognized by L-selectin. The conformation of the branched trisaccharide portion in the bound state remains close to the conformation of the free ligand. In the E-, P-, and L-selectin complexes the GalH4 proton is in the vicinity of protein aromatic protons, most likely Tyr94 and/or Tyr48.