Tumor necrosis factor receptor (TNF‐R) and APO‐1/Fas (CD95) are members of the tumor necrosis factor/nerve growth factor receptor superfamily involved in various forms of physiological cell death. Due to the structural homology between these receptors and their ligands, it has been suggested that APO‐1/Fas and TNF‐R kill cells by similar mechanisms. Here, we compared the killing pathways mediated by each receptor molecule in TNF‐sensitive L929 cells stably transfected with APO‐1/Fas cDNA. Morphological analysis revealed that TNF‐induced cell death resembles necrosis, while APO‐1/Fas‐mediated cell killing shows an apoptotic pattern, evident by the appearance of membrane blebbing, nuclear condensation and non‐random DNA degradation. Studies with inhibitors of several intracellular pathways further demonstrate that the mechanisms of TNF‐ and APO‐1/Fas‐mediated cell killing are substantially different. TNF cytotoxicity is mediated by reactive oxygen intermediates generated during mitochondrial respiration. However, these mediators are not involved in APO‐1/Fas‐mediated cell death as neither mitochondrial inhibitors nor antioxidants exert a protecting effect. Moreover, several inhibitors of calcium metabolism, ADP ribosylation and phospholipase action suppress TNF cytotoxicity, but not APO‐1/Fas‐mediated apoptosis. Additional differences between the two molecules were observed at the transcriptional level. Whereas transcription factor NF‐kappa B was readily activated by TNF, activation was not induced by triggering APO‐1/Fas. These data suggest that the two molecules, though structurally related, utilize distinct signal transduction pathways, even in a single cell type. Hence, cells may undergo different programs of cell death depending on the activating stimulus.