L-selectin is a cell surface adhesion receptor that mediates leukocyte rolling along vascular endothelium at sites of inflammation and lymphocyte attachment to endothelial cells within peripheral lymphoid tissues. Since ligation of L-selectin through its ligand recognition region may mimic physiologic ligand binding, a new panel of mAbs that engaged a conserved ligand-binding region within the lectin domains of human, mouse, and rat L-selectin were generated using L-selectin-deficient mice. Indeed, appropriate ligation of L-selectin generated transmembrane signals that resulted in immediate intercellular adhesion following cell-cell contact of lymphocytes, neutrophils, and L-selectin cDNA-transfected cells. Ab binding to only some epitopes within the lectin domain of L-selectin induced adhesion, while mAb binding to numerous other epitopes or other domains of L-selectin had no effect. PPME (yeast polyphosphomonoester core polysaccharide), a complex carbohydrate that mimics the natural L-selectin ligand, also induced potent intercellular adhesion. The induction of intercellular adhesion required cellular energy, an intact cytoskeleton, and cytoplasmic kinase activity as well as the membrane proximal and cytoplasmic domains of L-selectin. Therefore, ligation of L-selectin through specific ligand-binding epitopes identified by the mAbs generated in this study can generate transmembrane signals. Signaling through L-selectin may enhance leukocyte-endothelial cell interactions by serving as an activation/priming step during rolling, thereby promoting subsequent firm cell-cell adhesion in the presence of inflammatory mediators.