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This report describes a rapid, reproducible in vitro bioassay to quantitate the cytotoxic activity of human tumor necrosis factor-α using a human rather than murine cell line in the absence of metabolic inhibitors. The target cells are BT-20 (breast carcinoma) cultured at 39°C in the presence of recombinant human tumor necrosis factor-a (rHuTNF-α) in 96-well microtiter plates for 2 days. Cytotoxicity is measured by the crystal violet dye uptake of the remaining viable cells. This bioassay is sensitive to 1.5 ng/ml of rHuTNF-α, with an assay range to 130 ng/ml. Samples spiked into human plasma are measurable from 0.5 to 150 ng/ml. The specificity of this cytotoxic effect on the BT-20 cell line was demonstrated using rHuTNF-α neutralizing antibodies. A panel of cytokines including interferons, interleukins, and tumor necrosis factors was also analyzed using this assay system. Of the cytokines assayed, only recombinant murine tumor necrosis factor-α and recombinant human tumor necrosis factor-β demonstrated measurable cytotoxic activity when assayed independently, while recombinant human interferon-7 was the only cytokine to demonstrate greater than additive activity in combination with rHuTNF-α. The simplicity and reproducibility of this assay on a human cell line makes it useful for the routine determination of the biological activity of human tumor necrosis factor-α.