Oligonucleotide aptamers generated against purified LS‐Rg, a human L‐selectin/IgG fusion protein, bound human CD62L‐positive leukocytes. FACS analysis of lymphocytes or neutrophils stained with fluorescently labeled aptamers indicated specificity and sensitivity for cellular L‐selectin similar to that observed with anti–L‐selectin antibody. Univalent aptamers were compared to bivalent aptamers as well as to the anti‐adhesion, anti–L‐selectin antibody DREG56. Equilibrium and kinetic binding experiments were performed to examine the affinity and kinetic binding parameters of L‐selectin aptamers to evaluate their binding to CD62L‐positive leukocytes and to test their potential as L‐selectin antagonists. Binding experiments indicated that bivalent aptamers approached the affinity and the dissociation rate of bivalent antibody, and preferentially recognized cellular compared to soluble L‐selectin, a potentially useful distinction in vivo. Anti–L‐selectin aptamers also inhibited L‐selectin dependent self‐adhesion of neutrophils suggesting that in vitro univalent and bivalent aptamers provided anti‐adhesion activity similar to that observed with blocking antibody and indicated a direct blocking mechanism of action during inhibition of L‐selectin–dependent trafficking of lymphocytes observed in vivo. Cytometry 33: 394–405, 1998. © 1998 Wiley‐Liss, Inc.