A novel approach to the isolation of positive mediators of programmed cell death, based on random inactivation of genes by expression of anti sense RNAs, was employed to identify mediators of interferon-gamma-induced apoptosis. One of the several genes identified is DAP3, which codes for a 46 kDa protein with a potential nucleotide-binding motif. Structure-function studies of the protein indicate that the intact full-length protein is required for its ability to induce apoptosis when overexpressed. The N-terminal 230 amino acids, on the other hand, act in a dominant-negative fashion. Both of these functions are dependent on the integrity of the nucleotide binding motif. Expression of anti-sense DAP3 RNA and of the dominant interfering form of DAP3 both protected cells from apoptosis induced by activation of Fas and tumor necrosis factor alpha (TNF-alpha) receptors. Thus, DAP3 is implicated as a positive mediator of these death-inducing stimuli. It functions downstream of the receptor signaling complex and its death promoting effects depend on caspase activity. In the nematode Caenorhabditis elegans, a potential homolog of DAP3 showing 35% identity and 64% similarity to the human protein was isolated. Overexpression of the nematode DAP3 cDNA in mammalian cells induced cell death, indicating that the protein is conserved at the functional level as well as the structural level.