Induced shedding of the p55 tumor necrosis factor receptor (p55-R) was previously shown to be independent of the amino acid sequence properties of the intracellular domain of this receptor. We now find it also independent of the sequence properties of the transmembrane domain and of the cysteine-rich region that constitutes most of the extracellular domain of the receptor. The shedding is shown to depend solely on the sequence properties of a small region within the spacer that links the cysteine-rich region in the extracellular domain to the transmembrane domain. Detailed tests of effects of mutations in the spacer on the shedding indicate that the process is independent of the amino acid side-chain identity in this region except for a limited dependence on the identity of 1 residue (Val-173), located downstream to the putative major cleavage site of the receptor. It is strongly affected, however, by some mutations that seem to change the conformation of the spacer region. These findings suggest that a short amino acid sequence in the p55-R is essential and sufficient for its shedding and that the shedding is mediated either by a protease with limited sequence specificity or by several different proteases that recognize different amino acid sequences, yet it strictly depends on some conformational features of the cleavage region in the receptor.