Prolactin (PRL) has been shown to participate in lymphocyte activation. In particular, the constitutive natural killer (NK) and the lymphokine-activated killer (LAK) cytotoxicity of CD56+ CD16+ cells is increased by its physiological to supraphysiological concentrations. As PRL has been shown to up-regulate the production of interferon-γ (IFN-γ) by peripheral blood mononuclear cells, we studied its effect on IFN-γ production by NK cells as a possible mechanism of autocrine activation of cytotoxicity. Released and intracellular IFN-γ, as well as IFN-γ mRNA expression, were increased by pituitary and recombinant human PRL, which stimulated optimal NK and LAK cytotoxicity. Treatment with blocking anti-IFN-γ monoclonal antibody (mAb) selectively affected PRL-increased killing of K562 targets, demonstrating that PRL-mediated enhancement of spontaneous cytotoxicity depends, at least in part, on up-regulation of IFN-γ.