The ectodomain of the human transferrin receptor (TfR) is released as soluble TfR into the blood by cleavage within a stalk. The major cleavage site is located C-terminally of Arg-100; alternative cleavage sites are also present. Since the cleavage process is still unclear, we looked for proteases involved in TfR ectodomain release. In the supernatant of U937 histiocytic cells we detected alternatively cleaved TfR (at Glu-110). In membrane fractions of these cells we identified two distinct proteolytic activities responsible for TfR cleavage within the stalk at either Val-108 or Lys-95. Both activities could be inhibited by serine protease inhibitors, but not by inhibitors of any other class of proteases. Protein purification yielded a 28 kDa protein that generated the Val-108 terminus. The protease activity could be ascribed to neutrophil elastase according to the substrate specificity determined by amino acid substitution analysis of synthetic peptides, an inhibitor profile, the size of the protease and the use of specific antibodies. The results of analogous experiments suggest that the second activity is represented by another serine protease, cathepsin G. Thus, membrane-associated forms of neutrophil elastase and cathepsin G may be involved in alternative TfR shedding in U937 cells.