The addition of PHA [phytohemagglutinin] to C3H mouse spleen cells in tissue culture led to the production of lymphotoxin (LT). Cytotoxicity was assayed by addition of the culture fluids to syngeneic target cells [C3H mouse embryo tumor cells] labeled with 3H-thymidine; after an incubation period of 72 h the amount of radioactivity released into the supernatant was measured. The LT activity in unfractionated culture fluids survived lyophilization, remained unchanged for many wk at 4.degree. C, and progressively decreased on heating at 56.degree. C for periods from 1-9 h. Based on the G-200 Sephadex distribution coefficients for several preparations, the MW of mouse lymphotoxin was about 41,000 daltons. Lymphotoxin from 3 different spleen cell production runs was recovered from isoelectric focusing columns in sharply focused peaks, the pH of which ranged from 4.4-4.8.