A sensitive, reproducible in vitro bioassay is described for quantitating the cytolytic activity of tumor necrosis factor (TNF). The assay target cells, murine connective tissue L-M, are propagated and the assay performed under serum-free conditions. The quantitation of cytolytic activity is based on the ability of TNF to lyse L-M cells in the presence of actinomycin D, as measured by crystal violet dye uptake of residual viable cells. The assay is sensitive to 88 pg/ml TNF-alpha. The simplicity of the culture medium combined with high sensitivity and low variability make this a particularly well-suited bioassay for routine detection of TNF cytolytic activity.