Tumor necrosis factor (TNF) is an important mediator of many biological processes. This study sought to develop a sensitive bioassay allowing quantitative determination of TNF. The assay is based on uptake of the membrane impermeant dye, propidium iodide, by L929 cells when their viability is lost. L929 cells were plated on 96-well plates and cultured overnight in Eagle's minimum essential medium containing 10% horse serum. Propidium iodide (30 microM) was added, and fluorescence was monitored with a multiwell fluorescence scanner using 560-nm excitation and 645-nm emission filters. Human recombinant TNF caused a dose- and time-dependent increase in fluorescence, indicating onset of cell death. Cycloheximide (50 micrograms/ml) and actinomycin D (5 micrograms/ml) sensitized L929 cells to TNF-induced killing. By measuring cell killing early (12 h) and late (30 h), responses proportional to TNF concentration were obtained over a dynamic range of 1 to 5000 pg/ml. These findings serve as the basis for a simple TNF assay that is easy to perform and sensitive over a very broad range of TNF concentrations.