L-selectin mediates the initial tethering and subsequent rolling of leucocytes along luminal walls of inflamed venules. TACE [TNFalpha (tumour necrosis factor alpha)-converting enzyme] is responsible for cleaving the membrane-proximal extracellular domain of L-selectin (also known as shedding), which reduces the efficiency of leucocyte recruitment to sites of inflammation. Many reports have highlighted roles for PKC (protein kinase C) and p38 MAPK (mitogen-activated protein kinase) in promoting L-selectin shedding with little insight into the mechanism involved. By using PMA and the phosphatase inhibitors cantharidin and calyculin A, we could selectively activate PKC or p38 MAPK respectively to promote TACE-dependent shedding of L-selectin. Interestingly, the intracellular mechanisms leading to the shedding event differed dramatically. For example, regulatory elements within the L-selectin cytoplasmic tail, such as ERM (ezrin/radixin/moesin)-binding and serine residues, were important for PKC- but not p38 MAPK-dependent shedding. Also, increased and sustained cell surface levels of TACE, and phosphorylation of its cytoplasmic tail (a hallmark of TACE activation), occurred in lymphocytes and monocytes following p38 MAPK activation. Finally, we showed that TNFalpha-induced shedding of L-selectin in monocytes was strikingly similar to cantharidin-induced shedding and suggest that this newly characterized mechanism could be physiologically relevant in inflammatory cells.