Human monocytes express both Fas and Fas ligand (FasL) on the cell surface, and the interaction of these molecules induces spontaneous apoptosis. In this report we present a study of monocytic cells by FACS and confocal microscopy using anti-FasL Abs that reveals high levels of preformed FasL within the intracellular compartment. Further analysis by immunoblotting of cell cytoplasmic proteins confirmed the presence of a 37-kDa protein recognized by anti-FasL Abs. Stimulation of the monocytic cells with immune complexes, PHA, or superantigen gave rise to the rapid release of soluble FasL from within the cells. The presence of high levels of FasL within human monocytes suggests that, upon stimulation, the cells can rapidly translocate intracellular FasL to the cell surface and release it into the extracellular milieu. These findings indicate a novel mechanism for monocytes to respond rapidly to environmental changes, resulting in the release of active, soluble FasL.