Supernatant fluids of mitogen-activated human tonsil lymphocytes contain large amounts of a factor toxic to mouse L cells. This substance, with a m.w. of 80,000 ±5,000 daltons, is called α-lymphotoxin (α-LT), to differentiate it from another toxin elaborated by mitogen-activated human blood lymphocytes, called β-lymphotoxin (β-LT), which differs from α-LT in size (45,000 ±5,000 daltons), antigenicity, and stability. Further purification of α-LT by sequential phosphocellulose and DEAE-cellulose chromatography and polyacrylamide gel electrophoresis (PAGE) identifies a series of cytotoxins differing in ion exchange characteristics and electrophoretic mobilities. The three PAGE fractions (PAGE Ia, Ib and II), recovered in 2, 4.6, and 21% yield from the starting serum-free culture supernatant, represent purifications of 24-, 24- and 1851-fold, respectively. Each cytotoxic fraction has a ribonuclease activity. Comparison of RNase and mouse L cell cytotoxic activities of the three α-LT fractions shows that both activities for all three fractions have a similar temperature stability pattern and that both are similarly inhibited by DNA, single strand forms better than double strands, by glycerol in 5 to 20% concentration, and by protein denaturing reagents. These observations suggest, but do not prove, that mouse L cell toxicity and RNase activity are mediated by the same substance, which appears to occur in multiple or isozymic forms.