A standardized microculture system has been developed to assess the ability of lymphocytes to secrete leukocyte migration inhibitory factor (LMIF) in response to the nonspecific mitogen concanavalin(Con A). LMIF-rich supernates collected from stimulated lymphocytes cultured in plastic microtiter plates are assayed by pulse exposure of purified human granulocytes and inhibition of their migration in agarose medium. LMIF activity in this system is suppressed by the protein synthesis inhibitor puromycin, but not by inhibition of lymphocyte proliferation by irradiation. It is demonstrated that normal lymphocytes stimulated with mitogen elaborate LMIF activity, while lymphocytes from malignant lymphoma patients are frequently unable to produce it. Thus, mitogen-induced mediator production may be a useful parameter in further characterization of primary and secondary immunodeficiencies.