Galectin-3, a lactose-binding mammalian lectin that is secreted from activated macrophages, basophils, and mast cells, was investigated with respect to its ability to activate the human neutrophil NADPH-oxidase. The galectin-3-induced activity was determined with in vivo exudated cells (obtained from a skin chamber) and compared with that of peripheral blood neutrophils. Galectin-3 was found to be a potent activator of the NADPH-oxidase only in exudated neutrophils and the binding of galectin-3 to the surface of these cells was increased compared with peripheral blood cells. Different in vitro priming protocols resulting in degranulation were used to mimic the exudation process in terms of increasing the receptor exposure on the cell surface. Galectin-3 could induce an oxidative response similar to that in exudated cells only after a significant amount of the intracellular organelles had been mobilized. This increase in oxidative response was paralleled by an increased binding of galectin-3 to the surface of the cells. The major conclusion of the study is that galectin-3 is a potent stimulus of the neutrophil respiratory burst, provided that the cells have first experienced an extravasation process. The results also imply that the neutrophil response to galectin-3 could be mediated through receptors mobilized from intracellular granules, and we report the presence of galectin-3-binding proteins in such organelles.