Unlike other tumor necrosis factor family members, the cytotoxic ligand tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)/Apo-2L contains an unpaired cysteine residue (Cys 230 ) in its receptor-binding domain. Here we show that the biological activity of both soluble recombinant TRAIL and cell-associated, full-length TRAIL is critically dependent on the presence of Cys 230 . Mutation of Cys 230 to alanine or serine strongly affected its ability to kill target cells. Binding to its receptors was decreased by at least 200-fold, and the stability of its trimeric structure was reduced. In recombinant TRAIL, Cys 230 was found engaged either in interchain disulfide bridge formation, resulting in poorly active TRAIL, or in the chelation of one zinc atom per TRAIL trimer in the active, pro-apoptotic form of TRAIL.Ligands and receptors of the tumor necrosis factor (TNF) 1 family function to trigger cell growth, differentiation, activation, and apoptosis and are mainly implicated in the maintenance and function of the immune system (1). The TNF-related apoptosis-inducing ligand (TRAIL/Apo-2L) displays cytotoxic activity toward a number of transformed cell lines but not against most primary cells, suggesting that it may be a useful anti-tumor agent (2, 3). Recent reports demonstrating that TRAIL is able to counteract progression of human tumor xenografts in a murine model support this hypothesis (4, 5).There are several receptors for TRAIL that all belong to the TNF receptor family. These include TRAIL-R1/DR4, TRAIL-R2/DR5, TRAIL-R3/DcR1, and TRAIL-R4/DcR2, which display high sequence homology in their extracellular domains and have TRAIL as their only known ligand. Osteoprotegerin (OPG), however, belongs to a distinct subfamily and binds to both TRANCE/RANKL/OPGL and TRAIL. The widely expressed TRAIL-R1 and TRAIL-R2 contain an intracellular death domain and can signal apoptosis; the remaining receptors are believed to function as decoys and either do not contain a functional death domain (TRAIL-R4), have a glycolipid anchor (TRAIL-R3), or exist in a soluble form (osteoprotegerin) (for a review on TRAILRs, see Ref. 6).The extracellular domains of TNF receptor family members have an elongated shape stabilized by multiple disulfide bridges, which is reflected at the level of the primary structure by a characteristic pattern of Cys residues (7). In contrast, ligands are formed of anti-parallel -sheets folding into the so-called "jellyroll" structure, which in some cases is stabilized by a single disulfide bridge (8,9). TRAIL is remarkable in that it contains an unpaired Cys residue (Cys 230 ) at the corresponding position where some other ligands have a disulfide bridge. TRAIL, alone or in complex with TRAIL-R2, has been crystallized recently by three different groups (10 -12). Although Cys 230 appears to be important in the organization of the different structures, it has been found either (a) as a free Cys residue, (b) involved in disulfide bridge formation with Cys 230 of another monomer (and as a free Cys ...