The localization of the adhesion protein L‐selectin in human neutrophils was determined by sub‐cellular fractionation and immunoelectron microscopy and compared with the localization of Mac‐1 (α _m β _z ) and alkaline phosphatase, the marker for secretory vesicles. L‐selectin was found to be localized exclusively on the plasma membrane of unstimulated cells and also of stimulated cells, although markedly diminished. This was in contrast to Mac‐1, which was also localized in secretory vesicles and in specific/gelatinase granules as shown previously [Sengeiov, H., et al. J. Clin. Invest. (1993) 92, 1467–1476]. Stimulation of neutrophils with inflammatory mediators such as tumor necrosis factor (TNF), platelet‐activating factor (PAF), or f‐Met‐Leu‐Phe (fMLP), induced parallel up‐regulation of the surface membrane content of alkaline phosphatase and Mac‐1 and down‐regulation of L‐selectin, as evidenced by flow cytometry. Preimbedding immunoelectron microscopy confirmed that L‐selectin was present mainly on tips of microvilli in unstimulated cells and showed that alkaline phosphatase and Mac‐1 were randomly distributed on the surface membrane of fMLP‐stimulated cells. These studies indicate that the transition of neutrophils from L‐selectin‐presenting cells to Mac‐l‐presenting cells induced by inflammatory mediators is mediated by incorporation of secretory vesicle membrane, rich in Mac‐1 and devoid of L‐selectin, into the plasma membrane. J. Leukoc. Biol. 56: 80–87; 1994.