A soluble form of the human transferrin receptor (TfR) resulting from proteolytic cleavage at Arg 100 has been measured in human blood. In tissue culture cells elimination of the O-linked carbohydrate at Thr 104, four amino acids from the cleavage site, results in enhanced cleavage of the TfR (Rutledge et al., 1994, Blood, 83:580-586). In the present set of studies, the influence of amino acid substitution and the composition of the oligosaccharide at amino acid 104 on the cleavage of the TfR was examined. Site-directed mutagenesis was used to generate six different amino acids at position 104 which varied in size and charge. Measurement of the soluble TfR in the conditioned medium of the transfected cells of each mutant TfR showed that the large and charged side chains inhibited TfR cleavage the most. Otherwise the properties of the mutant TfRs were indistinguishable from the wild-type TfR in that the affinity of transferrin for these receptors, the extent of disulfide bond formation of the TfRs, and the proportion of TfRs at the cell surface were similar to that of the wild-type TfR. Removal of the sialic acid component of the carbohydrate from wild-type TfR by treatment of live cells with neuraminidase enhances TfR cleavage. Expression of wild-type TfR in CHO IdlD cells (a glycosylation defective cell line) also shows enhanced cleavage under conditions that produce truncated or no O-linked carbohydrates. Treatment of IdlD cells with neuraminidase reveals that the sialic acid of the O-linked carbohydrate protects against TfR cleavage, whereas the core sugars Gal-NAc and Gal do not protect as much. These results show that the terminal charged sialic acid residues are important for protection from proteolytic cleavage and suggest that cleavage could be regulated in the cell by removal of all or part of the carbohydrate.