To determine the region of human tumor necrosis factor-alpha (TNF-α), essential for cytotoxic activity against mouse L-M cells, single amino-acid-substituted TNF-α mutant proteins (muteins) were produced in Escherichia coli by protein engineering techniques. An expression plasmid for TNF-α was mutagenized by passage through an E.coli mutD5 mutator strain and by oligonucleotide-directed mutagenesis. Approximately 100 single amino-acid-substituted TNF-α muteins were produced and assayed for cytotoxic activity. The cytotoxic activities of purified TNF-α muteins, e.g. TNF-31T, -32Y, -82D, -85H, -115L, -141Y, -144K and -146E, were < 1% of that of parent TNF-α. These results indicate that the integrity of at least four distinct regions of the TNF-α molecule is required for full biological activity. These regions are designated as follows: region I, from position 30 to 32; region II, from position 82 to 89; region III, from position 115 to 117; region FV, from position 141 to 146. In addition, TNF-141Y could not completely compete with parent TNF-α for binding to the receptor. This demonstrates that region IV, and at least aspartk acid at position 141, must be involved in the TNF receptor binding site.