The activation of pure populations of cloned peritoneal macrophages for tumor cell cytotoxicity by Con A was demonstrated using a recently developed tumor cell clonogenic assay. Cloned macrophages were rendered cytotoxic by Con A at concentrations above 20 micrograms/ml. The tumor cell cytotoxicity was caused mainly by the tumoricidal activity of the Con A-activated cloned macrophages. Increasing The Con A-activation time from 24 hours to 48 hours and 72 hours heightened the cytotoxic activity of cloned macrophages. Cloned macrophages incubated with con A for only 2 hours possessed no cytotoxic effect. Culture fluid from cultures of activated macrophages exerted no tumor cell cytotoxicity. Alpha-methyl-D-mannoside, a specific receptor-binding inhibitor for Con A, was capable of blocking the activation of macrophages for cytotoxicity at 0.01 M concentration.