Blood-borne lymphocyte trafficking to peripheral lymph nodes (PLNs) depends on the successful initiation of rolling interactions mediated by L-selectin binding to sialomucin ligands in high endothelial venules (HEVs). Biochemical analysis of purified L-selectin ligands has identified posttranslational modifications mediated by Core2GlcNAcT-I and high endothelial cell GlcNAc-6-sulfotransferase (HECGlcNAc6ST). Consequently, lymphocyte migration to PLNs of C2GlcNAcT-I(-/-) and HEC-GlcNAc6ST(-/-) mice was reduced; however, B-cell homing was more severely compromised than T-cell migration. Accordingly, intravital microscopy (IVM) of PLN HEVs revealed a defect in B-cell tethering and increased rolling velocity (V(roll)) in C2GlcNAcT-I(-/-) mice that was more pronounced than it was for T cells. By contrast, B- and T-cell tethering was normal in HEC-GlcNAc6ST(-/-) HEVs, but V(roll) was accelerated, especially for B cells. The increased sensitivity of B cells to glycan deficiencies was caused by lower expression levels of L-selectin; L-selectin(+/-) T cells expressing L-selectin levels equivalent to those of B cells exhibited intravascular behavior similar to that of B cells. These results demonstrate distinct functions for C2GlcNAcT-I and HEC-GlcNAc6ST in the differential elaboration of HEV glycoproteins that set a threshold for the amount of L-selectin needed for lymphocyte homing.