The adhesion molecule L-selectin expressed on most leukocytes mediates tethering and rolling of leukocytes on activated endothelia and initiates the extravasation of leukocytes into inflamed tissues. Recombinant L-selectin-Fcγ is widely used both as a tool to study this key step of inflammation and as an anti-inflammatory compound in animal models of inflammation. Since previous studies on cellular L-selectin have indicated that glycosylation influences adhesive interactions of the adhesion molecule, we have examined whether the binding activity of L-selectin-Fcγ is affected by sialylation. Different forms of recombinant human L-selectin-Fcγ were expressed in CHO and K-562 cells and were purified by affinity chromatography using Protein A-Sepharose. A hypersialylated form of L-selectin-Fcγ was generated by culturing cells in the presence of 5 mM N-acetyl-beta-d-mannosamine, while a desialylated variant was obtained by treatment of purified L-selectin-Fcγ with neuraminidase. Binding activity to the synthetic biligand SiaLex-PAA-sTyr was measured by surface plasmon resonance (SPR) technology. While hypersialylated L-selectin-Fcγ showed decreased binding activity, desialylation elevated L-selectin-Fcγ binding to SiaLex-PAA-sTyr. The data show that sialylation of L-selectin-Fcγ reduces binding activity to ligand epitopes containing sialyl Lewis x and sulfated tyrosine residues. For the production of biologically active L-selectin-Fcγ conditions should be chosen that favor the generation of non-sialylated or of scarcely sialylated forms of the recombinant glycoprotein.