Selectins are carbohydrate-binding adhesion molecules that play important roles in control of leukocyte traffic. Glycosyltransferases involved in selectin ligand biosynthesis include the α1,3-fucosyltransferases FucT-VII and FucT-IV, one or more sialyltransferases, and at least one O-linked branching enzyme. Previous studies have shown that core 2 β1-6-N-glucosaminyltransferase (C2GlcNAcT-I; EC 2.4.1.102) is required for functional modification of PSGL-1, the leukocyte P-selectin ligand, but have been ambiguous on whether this enzyme is involved in E-selectin ligand formation. Using an attachment and rolling assay under defined shear flow in vitro, this study shows that C2GlcNAcT-I⁻ lymphoid cells stably transfected with FucT-VII complementary DNA attach and roll well on E-selectin at 1.5 dynes/cm.² Further, attachment and rolling on P-selectin of neutrophils is sharply reduced and that of short- term polarized Th1 cells is virtually abolished, with leukocytes from C2GlcNAcT-I^−/− mice. In contrast, both neutrophils and Th1 cells from C2GlcNAcT-I^−/− mice attach and roll as well as wild-type cells on E-selectin. These results show that C2GlcNAcT-I is selectively required for biosynthesis of ligands for P-selectin, but is not essential for at least some E-selectin ligands. Distinct requirements for C2GlcNAcT-I in the formation of ligands for E-selectin versus P-selectin represents a novel level of regulation of expression of selectin ligands and lymphocyte traffic.