During in vitro chemotaxis, human embryonic fibroblasts migrate toward the leukotriene B4 (LTB4) contained in ionophore-induced human mononuclear-cell supernatants, but they do not migrate toward the LTB4 contained in ionophore-induced neutrophil supernatants. We further analyzed and characterized this inhibitory effect. The inhibitor was found to be present in stimulated, but not in unstimulated, neutrophil supernatants. The inhibitor was also shown to be heat labile, to interfere with the chemotaxis of the tumor cell lines HT 1080 and L 929, and to be effective during chemotaxis stimulated by LTB4, conditioned medium, or fibronectin. At Sephadex-G-200 chromatography, the inhibitor eluted in a region corresponding to a molecular mass of 16,000 daltons. Preincubation experiments showed that its mechanism of action is not cell directed, and it had no effect on random migration, cell spreading, and cell attachment. Furthermore, the inhibitor does not interact with the binding of fibronectin to its specific antibody; thus, an interaction of the inhibitor with nonspecific sites which are common to several chemotactic factors must be postulated instead. The biological role of the inhibitor may be related to the regulation of cell migration during wound repair.