Recent studies have variably reported that tumour necrosis factor �� (TNF-��) induces either necrosis or apoptosis in L929 cells. This study was undertaken to better characterize the mode of death induced in L929 cells by this agent. We determined the effects of exposure to TNF-�� and other cytotoxic agents on cell size and morphology, cell membrane permeability, exposure of phosphatidylserine at the cell surface, nuclear morphology and fragmentation of DNA. Our results suggest that L929 cells treated with TNF-�� alone show nuclear changes and a pattern of DNA fragmentation that are atypical of apoptosis. In contrast, our results demonstrate that, when augmented with actinomycin D, TNF-�� induces classical apoptosis in L929 cells. We also provide the first report that, in L929 cells, staurosporine induces classical apoptosis and colchicine induces a form of apoptosis lacking internucleosomal DNA fragmentation. Previous studies of TNF-��-induced death in L929 cells relied on measurements of only one or two parameters to define the mode of death. Overall, our results suggest that in future cellular or biochemical studies of the effects of TNF-�� on L929 cells it will be prudent to characterize the mode of death in each case using a multi-parameter approach, as done here.