E-selectin and P-selectin are two closely related vascular cell adhesion proteins. Each selectin has an amino-terminal C-type lectin domain that is thought to possess the carbohydrate binding site that binds the sialylated Lewisx antigen (sLex or CD15s) (Neu5Acalpha2-3Galbeta1-4(Fucalpha1-3)GlcNAc). In addition to the sLex carbohydrate, P-selectin binds sulfated proteoglycan, 3-sulfated galactosyl ceramide (sulfatide), and heparin. Both E- and P-selectin have an EGF-like (EGF) domain that is immediately adjacent to and COOH-terminal to the lectin domain. We report that mutagenic substitution of single amino acid residues in either the P- or E-selectin EGF domain can dramatically alter selectin binding to sLex, heparin, or sulfatide. Substitution of E- and P-selectin EGF domain residue Ser128 with an arginine results in E- and P-selectin proteins that have lost the requirement for alpha1-3-linked fucose and are thus able to bind to sialyllactosamine. A similar phenotype is reported for an E-selectin mutation within the lectin domain. Additionally, we have determined that conservative substitution of EGF domain residues 124 and 128 can alter E-selectin binding such that it is able to adhere to heparin or sulfatide and can reduce P-selectin adherence to these ligands. The distance between the substituted EGF domain amino acid residues and the primary carbohydrate binding site within the lectin domain and their relative positioning as determined by the three-dimensional crystal structure of the E-selectin lectin and EGF domains (Graves, B. J., Crowther, R. L., Chandran, C., Rumberger, J. B., Li, S., Huang, D.-S., Presky, D. H., Familletti, P. C., Wolitzky, B. A., and Burns, D. K. (1994) Nature 367, 532-538) suggest that there is little direct contact between the two domains. However, we report mutant binding characteristics which indicate that selectin oligosaccharide binding may be modulated by both domains and that wild-type E- and P-selectin/sLex binding interactions may be significantly different from those previously hypothesized.