Puromycin treatment of lymphocytes was used to develop a one-way test for leukocyte migration inhibitory factor (LMIF) production in the mixed lymphocyte culture (MLC) reaction. Lymphocytes incubated with this protein synthesis inhibitor induced a vigorous mediator production by nontreated allogeneic cells, being themselves unable to respond to stimulator cells. When puromycin-treated cells were stimulated with the mitogens PHA, ConA, or PWM, overall protein and DNA synthesis were significantly decreased with concomitant abolishment of LMIF production. Viability of stimulator lymphocytes was found to be essential for generation of the mediator in MLC reaction.