The production of macrophage migration inhibitory factor (MIF) and immune interferon (IF) by concanavalin A (Con A)-stimulated cultures of thymus, lymph node and spleen cells was investigated. It was found that all cultures produced MIF activity, whereas only spleen cells produced marked IF activity. The capacity to produce IF was found to be correlated with the macrophage content of a cell preparation as evidenced by staining for esterase-positive cells. Furthermore, column-purified spleen T cells produced MIF but no IF. Migration inhibition caused by residual mitogen could be ruled out. On the other hand, when macrophages grown from bone marrow cells were pre-exposed to supernatants of mitogen-stimulated lymphocytes, IF activity was released into freshly added medium while no significant MIF activity was found. IF was also found in supernatants of macrophage cultures after exposure to conventional inducers in vitro (polyinosinic-polycytidylic acid, Corynebacterium parvum) or in vivo (C. parvum), whereas no MIF was detected. An anti-Type I IF serum neutralized IF in supernatants from Con A-stimulated spleen cells but did not affect MIF in the same supernatants. This indicates that IF and MIF activity are associated with different molecules. It is, therefore, concluded that under the described conditions, IF and MIF are produced by different cells. T cells are the prime producers of MIF while IF is released by macrophages following induction by lymphokines.