Serum from Mycobacterium bovis BCG-infected mice treated with lipopolysaccharide was cytotoxic to tumor cells in vitro. Serum-induced cytotoxicity was estimated by measuring release of [3H]thymidine into culture supernatants of prelabeled tumor target cells. Serum from BCG-infected mice not treated with lipopolysaccharide or from uninfected mice treated with lipopolysaccharide was inactive. Moreover, although serum cytotoxic activity was evident with 10 syngeneic or allogeneic tumor cell lines, little or no effect was observed with normal embryonic fibroblast target cells. Maximal titers of serum cytotoxic activity were detected 14 days after BCG infection and 2 h after LPS treatment. Serum of BCT-infected, T-cell-deficient nude mice developed strong cytotoxic activity after LPS treatment; however, lipopolysaccharide-insensitive C3H/HeJ mice could produce this cytotoxic activity only after adoptive transfer with lipopolysaccharide-responsive C3H/HeN bone marrow. Physicochemical characterization of the serum cytotoxic activity revealed a heat-stable (56 degrees C, 30 min) entity with a molecular weight of about 60,000 and an isoelectric point at pH 4.8. Biological and physicochemical characteristics of this serum cytotoxic activity as defined by an in vitro assay were very similar to characteristics of tumor necrosis factor and suggest that this molecule may be a major effector mechanism for the antitumor actions of lipopolysaccharide.