The possibility that the activity of migration inhibitory factor (MIF) requires the presence of antigen in the culture of nonimmune peritoneal exudate cells has been suggested by several authors. In order to study such antigen-dependent MIF, we stimulated lymph node cell cultures from guinea pigs immunized with various DNP-protein conjugates with the immunizing antigen in vitro. The antigen was removed from MIF-containing supernatants by affinity chromatography on anti-DNP-agarose bead columns. The effluent material retained full MIF activity despite the absence of antigen. Addition of antigen did not increase the activity of these antigen-free supernatants. Moreover, no MIF activity could be subsequently eluted from the anti-DNP-agarose bead column by treatment with dilute acid, suggesting that no MIF, as antigen-MIF complex, had been removed by the anti-DNP column. In addition, DNP-protein-agarose bead conjugates stimulated lymphocytes to produce active MIF, although no antigen could be detected in these MIF-containing supernatants. Addition of antigen did not increase the activity of the supernatant. Thus, in these systems, no evidence for antigen-dependent MIF has been obtained.