Several cell-associated cytokines, such as interleukin-1 (IL-1) and tumor necrosis hctor, exist on the cell s d c e and are biologically active. Al- though extracellular IL-8, a potent chemotactic k t o r for pl.lmarily neutrophils, has been studied extensively, cell-associated IL-8 has barely been studied. In this study, we analyzed the intracellular and cell-surface IL-8 in human blood monocytes in vitro by using flow cytometry and predicted the biological activity of the cell-associated IL-8 i n vivo. After &ation with paraformaldehyde, mononuclear cells were divided into two subgroups. One subgroup was left untreated to study cell-associated antigens, and the other subgroup was permeabmed with saponin to detect intracellular antigens. In lipopolysaccharide (LPS)-stimulated monocytes, IL-8 was detected solely intracellularly, whereas both the intracellular and cell-surface IL-lP was detectable. In a time-course study, the intracellular IL-8 increased in response to LPS stimulation, but the cell-surface IL-8 was undetectable throughout the course. In an LPS-stimulated monocytic cell line, both ELISA and flow cytometry detected the quantitative change of the intracellular IL-8. The dissimilar localization between IL-8 and IL-lp within cells was codrmed by the immunohistochemical analysis. Ia summary, LPS stimulation induced a timedependent increase in intracellular but not cell-surh e IL-8 in monocytes. Thus, it is unlikely that the cell-associated IL.-8 is functioning physiologically. The semiquantitative flow cytometric procedure may be useful for simultaneous examination for cell-surface and intracellular cytokines. 0 1996 W i k y -J . i ~, I~c.