E- and P-selectin are inflammation-induced cell adhesion molecules that mediate leukocyte-endothelial cell and leukocyte-platelet interactions. Monoclonal antibodies (MoAbs) specific for either E-selectin or P-selectin are protective in several animal models of inflammatory disease. To generate an MoAb with broader therapeutic potential, MoAbs that bind to both E- and P-selectin were generated by immunization of mice with mouse pre-B cell lines transfected with human E- and P-selectin. Interestingly, although the only selection criterion was the ability to bind both E- and P-selectin, all three antibodies obtained efficiently block both E- and P-selectin-mediated functions. The inhibited functions include neutrophil or HL-60 cell binding to tumor necrosis factor-alpha-activated human umbilical vein endothelial cells, E- or P-selectin transfectant cell lines, and platelet-HL-60 rosetting. These antibodies, EP-5C7, EP-2C9, and EP-1D8, recognize the same or overlapping epitope within the lectin domains of E- and P-selectin. The data suggest that functionally important epitopes of homologous proteins can be targeted by selecting for antibodies with reactivity toward both proteins. Furthermore, a potent blocking antibody specific for both E- and P-selectin may provide a more effective and broadly useful reagent for treating acute and potentially certain chronic inflammatory conditions.