A variety of cytoplasmic and nuclear proteins can be modified on serine and threonine residues by O-linked beta-N-acetylglucosamine (O-GlcNAc), although the effects of this modification on protein and cellular functions are not completely defined. The sugar donor for the O-GlcNAc transferase that catalyzes this post-translational modification is UDP-N-acetylglucosamine (UDP-GlcNAc), a product of the hexosamine biosynthesis pathway (HBP). Here, the dynamics of the O-GlcNAc modification are examined in the physiological context of agonist-induced signal transduction using neutrophils. Formylated Met-Leu-Phe (fMLF) is shown to stimulate a rapid and transient increase in protein O-GlcNAcylation in both immunoblot and immunofluorescence imaging assays using O-GlcNAc-specific antibodies. In high performance liquid chromatography analyses of HBP metabolic activity, short term exposure to an exogenous substrate of the HBP, glucosamine (GlcNH(2)), leads to increased GlcNH(2) 6-phosphate and then UDP-GlcNAc levels. The GlcNH(2) treatments also increase O-GlcNAcylation and augment the aforementioned fMLF-associated increase. In functional assays, GlcNH(2) pre-treatment selectively augments fMLF-induced chemotaxis but has little effect on respiratory burst activity. Furthermore, augmenting levels of O-GlcNAc in the absence of agonist is sufficient to stimulate chemotaxis. These data demonstrate that neutrophils possess a functionally significant O-GlcNAcylation pathway that is robustly induced by stimulation with agonist. We propose that O-GlcNAcylation plays an important role in rapid and dynamic neutrophil signal transduction, especially with respect to chemotaxis.