Lymphocytes stimulated by Concanavalin A (Con A) as well as other mitogens release colony stimulating activity (CSA) which stimulated granulocyte and mononuclear cell formation from hematopoietic progenitor cells in vitro. Removal of Con A from lymphocyte cultures by the competitive inhibitor, α methyl mannoside, resulted in the cessation of CSA release regardless of the time the inhibitor was added to the cultures. However, removal of Con A after 6 hr of incubation had no effect on cellular DNA synthesis and lymphocyte transformation. In addition, vinblastine and cytosine arabinoside completely inhibited DNA synthesis but had no effect on CSA release. CSA release is dependent on an active energy process since it was completely and reversibly inhibited by the inhibitors of protein synthesis puromycin and cycloheximide. These results suggest that CSA release from stimulated lymphocytes requires continuous binding of mitogen and active protein synthesis but is not dependent on DNA synthesis and cell proliferation.