Trehalose dimycolate, a mycobacterial glycolipid also known as cord factor, retains some of the antitumor properties of the intact BCG. Murine macrophages incubated in vitro in the presence of trehalose dimycolate for 20 h at 37 degrees C released a factor which was cytotoxic for the L929 tumor cell line. Anti-tumor necrosis factor (TNF) blocked the cytotoxicity of trehalose dimycolate macrophage supernatant. Therefore the antitumor activity of trehalose dimycolate can be attributed to TNF released in the in vitro assay.