After exposure to mitogenic lectins in vitro, human mononuclear cells (95% lymphocytes) that had been prelabeled with [14C]arachidonic acid rapidly released a portion of their radioactivity in the medium. Most of the released radioactivity was demonstrated to be free arachidonic acid. Although other sources are not excluded, the most important source of cell-bound radioactivity in the release reaction appeared to be phosphatidylinositol, suggesting that at least part of the response is occurring through an increase in phospholipase A2 activity. By gas liquid chromatography, other fatty acids were also shown to be released, but there was considerable selectivity in the response for arachidonic acid. The response was dependent on the availability of free Ca++ in the medium and was enhanced by serum proteins and unlabeled arachidonic acid. Most of the response appeared to be from the the lymphocytes themselves rather than from contaminating cells. The rapid generation of free arachidonic acid in response to mitogenic lectins suggests a possible role for arachidonic acid metabolites in the activation process.