We describe here a cell-free ligand binding assay for E-selectin. The assay involves immobilizing soluble (E) selectin onto microtiter plates and incubating with ({ }^{125} \mathrm{I}) labeled carcinoembryonic antigen (CEA) which carries the tetrasaccharide sialyl Lewis (x\left(s^{*}\right)^{x}). The bound CEA is eluted by ethylene glycol bis( (\beta)-aminoethyl ether)- (N, N, N^{\prime}, N^{\prime}),-tetraacetic acid and monitored by radioactivity. The binding is abolished by preincubation of either CEA with an anti-sLe ({ }^{x}) antibody or (E)-selectin with a neutralizing anti-E-selectin antibody, indicating that this is an E-selectin/sLe ({ }^{x})-specific interaction. The binding is (\mathrm{Ca}^{+2}) and (\mathrm{pH}) dependent (the optimal pH at 7.0) and also requires sialic acid. Removal of sialic acid from CEA by neuraminidase digestion abrogates the binding. When the protein but not carbohydrate constituent of CEA was denatured by reduction and alkylation, the binding was partially eliminated, indicating that the interaction may also be dependent on either protein conformation or carbohydrate orientation. The assay is simple, sensitive, and comparable to other methods as indicated by an (\mathrm{IC}_{50}) of (1 \mathrm{~mm}) for (\mathrm{sLe}^{\mathrm{x}}). Thus, the assay should be useful for screening antagonists and studying E-selectin structure/function. ic 1994 Academic Press, Inc.