When cells of human macrophage-like cell line U937 are cultured in the presence of medium conditioned by mixed lymphocyte culture (MLC), PHA- or Con A-stimulated lymphocytes they demonstrate morphologic and functional characteristics of stimulation. The cells become larger, their surface more villous, and the cytoplasm has increased numbers of lysosomes and phagosomes. There is a marked (at least 10-fold) increase in antibody-dependent cellular cytotoxicity (ADCC) against both erythroid and tumor targets accompanied by increased expression of Fc receptors (FcR). There is also an augmentation of antibody-dependent phagocytosis. Medium conditioned by unstimulated lymphocytes, a B lymphoblastoid cell line or a fibroblast cell line produce very little stimulation of U937 cells. U937 cells gradually lose their increased ADCC capacity if they are washed free of conditioned medium (CM) but can be maintained in a stimulated state for long periods of time by culturing them in medium supplemented with PHA-CM at a concentration of 2%. Stimulation of macrophage-like cell line U937 may provide a useful model for further study of mechanisms of macrophage cytotoxicity and its modulation by products of activated lymphocytes.