Abstract Mucins are high molecular weight glycoproteins that provide a protective layer on epithelial surfaces and are involved in cell–cell interactions, signaling, and metastasis. The identification of several membrane-tethered mucins, including MUC1, MUC3, MUC4, and MUC12, has incited interest in the processing of these mucins and the mechanisms that govern their release from the cell surface. MUC1 consists of an extracellular subunit and a membrane-associated subunit. The two moieties are produced from a single precursor polypeptide by an early proteolytic cleavage event but remain associated throughout intracellular processing and transport to the cell surface. We identified the MUC1 proteolytic cleavage site and showed it to be identical in pancreas and colon cell lines and not to be influenced by the presence of heavily glycosylated tandem repeats. The MUC1 cleavage site shows homology with sequences in other cell-surface-associated proteins and may represent a common mechanism for processing of these molecules.