Expression of the L-selectin adhesion molecule can be rapidly down-modulated by regulated proteolysis at a membrane-proximal site. The L-selectin secretase has remained undefined, and the secretase activity is resistant to a broad panel of common protease inhibitors. We have developed an L-selectin-alkaline phosphatase reporter, consisting of the ectodomain of human placental alkaline phosphatase fused to the membrane-proximal cleavage, transmembrane, and cytoplasmic domains of L-selectin, to aid in the screening for L-selectin secretase inhibitors. A hydroxamic acid-based metalloprotease inhibitor, KD-IX-73-4, inhibited release of the L-selectin-alkaline phosphatase reporter in a dose-dependent manner. The hydroxamic acid-based peptide was also found to inhibit wild type L-selectin down-regulation from the surfaces of phorbol myristate acetate-activated peripheral blood lymphocytes and phytohemagglutinin-stimulated lymphoblasts. Analysis of the proteolytic cleavage fragments of L-selectin confirmed that KD-IX-73-4 inhibited L-selectin proteolysis. Lymphocyte L-selectin was not down-regulated when co-cultured with formylmethionylleucylphenylalanine-stimulated neutrophils, suggesting that the putative secretase acts in cis with the membrane-bound L-selectin. These results suggest that the L-selectin secretase activity may involve a cell surface, zinc-dependent metalloprotease, although L-selectin shedding is not affected by EDTA and may be related to the recently described activity involved in processing of membrane-bound TNF-α.