L-selectin mediates lymphocyte homing by facilitating lymphocyte adhesion to addressins expressed in the high endothelial venules (HEV) of secondary lymphoid organs. Peripheral node addressin recognized by the MECA-79 antibody is apparently part of the L-selectin ligand, but its chemical nature has been undefined. We now identify a sulfated extended core1 mucin-type O-glycan, Galβ1→4(sulfo→6)GlcNAcβ1→3Galβ1→3GalNAc, as the MECA-79 epitope. Molecular cloning of a HEV-expressed core1-β1,3-N-acetylglucosaminyltransferase (Core1-β3GlcNAcT) enabled the construction of the 6-sulfo sialyl Lewis x on extended core1 O-glycans, recapitulating the potent L-selectin-mediated, shear-dependent adhesion observed with novel L-selectin ligands derived from core2 β1,6-N-acetylglucosaminyltransferase-I null mice. These results identify Core1-β3GlcNAcT and its cognate extended core1 O-glycans as essential participants in the expression of the MECA-79-positive, HEV-specific L-selectin ligands required for lymphocyte homing.