TNF- converting enzyme (TACE, ADAM17) cleaves membrane-associated cytokines and receptors and thereby regulates inflammatory and immune events, as well as lung development and mucin production. For example, the TACE-mediated cleavage of the type II, 75-kDa TNF receptor (TNFR2) generates a soluble TNF-binding protein that modulates TNF bioactivity. TACE is synthesized as a latent pro-enzyme that is retained in an inactive state via an interaction between its pro-domain and catalytic domain. Although, the formation of an intramolecular bond between a cysteine in the pro-domain and a zinc atom in the catalytic site had been thought to mediate this inhibitory activity, it was recently reported that the cysteine switch motif is not required. Here, we hypothesized that the amino-terminus of the TACE pro-domain might contribute to the ability of the pro-domain to maintain TACE in an inactive state, independent of a cysteine switch mechanism. We synthesized a 37-amino acid peptide, corresponding to TACE amino acids 18 to 54 (N-TACE^18-54), and assessed whether it possessed TACE inhibitory activity. In an in vitro model assay system, N-TACE^18-54 attenuated TACE-catalyzed cleavage of a TNFR2:Fc substrate. Further, N-TACE^18-54 inhibited constitutive TNFR2 shedding from a human monocytic cell line by 42%. A 19-amino acid, leucine-rich domain, corresponding to TACE amino acids 30 to 48, demonstrated partial inhibitory activity. In summary, we have identified a sub-domain within the amino-terminus of the TACE pro-domain that attenuates TACE catalytic activity, independent of a cysteine switch mechanism, which provides new insight into the regulation of TACE enzymatic activity.