3H-Thymidine uptake of thymocytes from LPS-responder Balb/c mice in the presence of a submitogenic dose (0.5 microgram/ml) of con A in vitro was significantly enhanced by adding LPS (0.1 to 2.5 microgram/ml), while the uptake of thymocytes from LPS-nonresponder C3H/HeJ was not enhanced by LPS. However, "endotoxin soups," which were prepared from the supernatants of LPS-responder murine spleen cell cultures in the presence of LPS, clearly increased the incorporation of 3H-thymidine into C3H/HeJ thymocytes in the presence of this small amount of con A. The soup prepared from C3H/HeJ spleen cell cultures did not show any synergistic effect with con A. Even if the major histocompatibility between soup-producer cells and responder cells to con A was different, the soups were still effective. The active substance in the "endotoxin soups" was eluted through a Sepharose CL-4B column, and its molecular size was estimated to be about 20,000 daltons. The activity of the soups was destroyed by heating at 70 C for 30 min or at 80 C for 10 min. Digestion with trypsin destroyed the activity of the soups, but digestion with DNase or RNase did not. The role of the active substance in the soups in synergy with con A and its relation to the synergistic effect of con A and LPS are discussed.