Since infectious mononucleosis (IM) mononuclear cells spontaneously release the leukocyte (migration) inhibitory factor (LIF) in culture and since previous experiments in our laboratory have demonstrated that one or more substances with LIF-like activity are detectable in the serum (serum LIF) of young patients with heterophile-positive IM, an investigation was carried out to determine both the in vitro and in vivo LIF production in a series of IM patients sampled during the acute phase of the infection. The observation than only the unstimulated lymphocytes from serum LIF-positive IM patients released LIF in culture suggests that a single active product is involved in the in vitro and in vivo LIF activities observed in Epstein-Barr virus (EBV) infected subjects. The biological implications of this finding with respect to the underlying immunopathology of the EBV IM syndrome are discussed.