Siglec-7 is a CD33-related sialic acid-binding Ig-like lectin expressed strongly on NK cells, where it can function as an inhibitory receptor. Its sialic acid-binding activity on NK cells is masked by cis interactions with sialylated glycans, which are likely to be important for regulating the inhibitory function of Siglec-7, which exhibits an unusual preference for alpha2,8-linked disialic acids, a motif found in "b-series" gangliosides and some glycoproteins. To investigate the presence of alpha2,8-linked disialic acids on NK cells, T cells, monocytes, and B cells, we first analyzed their expression of all known alpha2,8-sialyltransferase genes by quantitative PCR. Unlike T cells, B cells, and monocytes, NK cells consistently expressed mRNA encoding ST8Sia VI, which creates alpha2,8-linked disialic acids on O-linked glycans of glycoproteins. All blood leukocytes expressed ST8Sia IV, implicated in polysialic acid synthesis, and NK cells variably expressed high levels of ST8Sia V mRNA required for GT3 expression. Two human IgM antibodies, Ha1 and Pi1, with specificity for the alpha2,8-disialyl motif reacted strongly with NK cells in a sialic acid-dependent manner and less strongly with T cells and monocytes. Antibody-induced clustering of Siglec-7 on NK cells resulted in partial colocalization with anti-Ha1. Finally, MALDI-TOF mass spectrometric analysis of isolated NK cell O-glycans revealed the presence of a peak at mass-to-charge ratio of 1619.4 mass units, corresponding to a putative alpha2,8-disialylated glycan. Together, these results suggest that NK cells are decorated with alpha2,8-disialic acid structures implicated in regulation of cellular activation via interactions with Siglec-7.