Syrian hamster lymphotoxin has three identified anticancer activities: (a) cytolytic activity measured as the release of 3H from [3H]thymidine-labeled murine alpha L-929 cells; (b) cytostatic activity assessed by the inhibition of cell colony formation of tumorigenic Syrian hamster cells; and (c) anticarcinogenic activity measured by inhibition of chemical carcinogen or radiation-induced morphological transformation of Syrian hamster fetal cells in vitro and in vivo. Lymphotoxin cytolytic, cytostatic, and anticarcinogenic activities have similar molecular weights of approximately 45,000 by ACA-44 Ultrogel chromatography. A three-step purification of (a) diafiltration and concentration, (b) column isoelectric focusing in a pH 4 to 6 gradient, and (c) ACA-44 Ultrogel column chromatography yields 25% recovery of lymphotoxin cytolytic and anticarcinogenic activities. The three-step purified lymphotoxin is free of detectable interferon, macrophage migration inhibitory factor, and mitogenic factors inducing hamster T-lymphocyte proliferation and is anticarcinogenic in vivo. The purified lymphotoxin had a specific anticarcinogenic activity of 58,500 units/mg of protein and a molar specific activity of 2,900 units/nmol. Although homogeneous in molecular size, the purified lymphotoxin is comprised of several molecular species. Cytolytic activity is associated with molecules having a single major isoelectric point of 5.0, whereas the cytostatic and anticarcinogenic activities comigrate with molecules with major isoelectric points of 5.0 and 4.6. This is the first evidence that the anticancer actions of lymphotoxin are properties of similarly sized but several different electrically charged molecules and that the several lymphotoxin species have differing anticancer activities.