L-selectin mediates leukocyte rolling on vascular endothelium at sites of inflammation and lymphocyte migration to peripheral lymph nodes. L-selectin is rapidly shed from the cell surface after leukocyte activation by a proteolytic mechanism that cleaves the receptor in a membrane proximal extracellular region. This process may allow rapid leukocyte detachment from the endothelial surface before entry into tissues. In this study, the structural requirements for regulation of human L-selectin endoproteolytic release were examined through analysis of chimeric selectin molecules and mutant L-selectin receptors. The use of chimeric selectins and a cytoplasmic tail truncation mutant demonstrated that the extracellular membrane-proximal 15-amino acid region of L-selectin is required for endoproteolytic release. The introduction of alanine-scanning mutations within this membrane-proximal region did not prevent endoproteolytic release, indicating that a specific amino acid motif was not an absolute requirement for cleavage. Furthermore, alterations within the putative primary cleavage site (K283-S284) resulted in either constitutive endoproteolytic release of the receptor or inhibition of cell activation-induced shedding to variable extents. The length of the membrane-proximal region was also critical since truncations of this region completely abolished endoproteolytic release. Thus, release of L-selectin is likely to be regulated by the generation of an appropriate tertiary conformation within the membrane-proximal region of the receptor which allows recognition by a membrane-bound endoprotease with relaxed sequence specificity that cleaves the receptor at a specific distance from the plasma membrane. These observations suggest a generalized protein-processing pathway involved in the endoproteolytic release of specific transmembrane proteins which harbor widely differing primary sequences at or neighboring their cleavage sites.