In order to define the active site(s) of human tumour necrosis factor (hTNF), we mutagenized its gene at random and directly screened the resulting population for loss of cytotoxic activity on L929 cells. Four biologically inactive mutant proteins (Arg32‐‐‐‐Trp, Leu36‐‐‐‐Phe, Ser86‐‐‐‐Phe and Ala84‐‐‐‐Val) behaved similar to the wild‐type in various physico‐chemical assays. The residues were positioned on a 3D structural model and were found to cluster together at the base of the molecule at each side of the groove that separates two monomers in the trimeric structure. A very conservative mutation at one of these sites (Ala84‐‐‐‐Val) almost completely abolished cytotoxic activity. Amino acid alterations in three other residues in close proximity to this receptor binding site were introduced: replacements at positions 29 and 146 clearly reduced cytotoxicity only when non‐conservative alterations were introduced (Leu29‐‐‐‐Ser and Glu146‐‐‐‐Lys), suggesting an indirect influence on the active site. However, a conservative mutation at position 91 (Val‐‐‐‐Ala) caused a significant drop (500‐fold) in bioactivity which suggests that Val91 may also play a direct role in receptor recognition. Our results favor a model in which each TNF molecule has three receptor‐interaction sites (between the three subunits), thus allowing signal transmission by receptor clustering.