Leukocyte traffic into lymph nodes and sites of inflammation is guided by L-selectin. Experiments performed in vitro and with gene-deleted mice suggest that CD34 recognizes L-selectin if decorated by 6-sulfo sialyl Lewis x (sLex) saccharides and the MECA-79 epitope. However, very little is known about glycosylation of human L-selectin ligands. We report here on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) profiles of N- and O-linked oligosaccharide fractions from human tonsillar endothelial CD34. All detected O-glycans were sialylated; some were also monosulfated or monosulfated and monofucosylated. If a given CD34-glycan may carry all requirements for L-selectin recognition, that is, both 6-sulfo-sLex and MECA-79 epitopes, only one O-glycan fraction, O-9, SA(2)Hex(3)HexNAc(3)- Fuc(1)(SO(3))(1), meets the criteria. A candidate structure is SAalpha2-3Galbeta1-4(Fucalpha1-3)(6-sulfo)GlcNAcbeta1-3Galbeta1-3(SAalpha2-3Galbeta1-4GlcNAcbeta1-6)GalNAc. However, if sulfo sLex glycans are supplemented with separate sulfated, nonfucosylated O-glycans, saccharides in O-6, O-8, or O-9, putatively carrying MECA-79 epitopes, could form multiglycan binding epitopes for L-selectin.