Deletion mutagenesis of the intracellular region of the 55 kd TNF receptor (TNF-Rl) identified an-80 amino acid domain near the C-terminus responsible for signaling cytotoxicity. This domain shows weak homology with the intracellular domain of Fas antigen, a transmembrane polypeptide that can also initiate a signal for cytotoxicity. Alanine-scanning mutagenesis of TNF-Rl confirmed that many of the amino acids consewed with Fas antigen are critical for the cytotoxic signal. This region of TNF-Rl-Fas homology is therefore likely to define a novel domain (death domain) that signals programed cell death. Mutations within the death domain of TNF-Rl also disrupted its ability to signal anti-viral activity and nitric oxide (NO) synthase induction. In addition, large deletions in the membrane-proximal half of the intracellular domain did not block signaling of cytotoxicity or anti-viral activity but did block induction of NO synthase.