Leukocyte capture and rolling are mediated by selectins expressed on leukocytes (L-selectin) and the vascular endothelium (P- and E-selectin). To investigate the role of core 2 β1-6-N-glucosaminyltransferase (C2GlcNAcT-I) for synthesis of functional selectin ligands in vivo, leukocyte rolling flux and velocity were studied in venules of untreated and tumor necrosis factor-α (TNFα)–pretreated autoperfused cremaster muscles of C2GlcNAcT-I–deficient (core 2^−/−) and littermate control mice. In untreated core 2^−/− mice, leukocyte rolling was dramatically reduced with markedly increased rolling velocities (81 ± 4 μm/s vs 44 ± 3 μm/s). The reduced rolling in core 2^−/− mice was due mainly to severely impaired binding of P-selectin to P-selectin glycoprotein ligand-1 (PSGL-1). Some rolling remained after blocking PSGL-1 in controls but not in core 2^−/− mice. In TNFα-pretreated mice, rolling was markedly reduced in core 2^−/− mice owing to impaired P-selectin– and E-selectin–mediated rolling. Rolling velocities in core 2^−/− mice treated with an E-selectin–blocking monoclonal antibody (59 ± 4 μm/s) were significantly higher than in controls (14 ± 1 μm/s), which provides further evidence for the severe impairment in P-selectin–mediated rolling. In conclusion, P-selectin ligands including PSGL-1 are largely C2GlcNAcT-I dependent. In addition, E-selectin–mediated rolling in vivo is partially dependent on the targeted C2GlcNAcT-I.