Recently, we developed an automated apparatus for rapid releasing of O-glycans from mucin-type glycoproteins and proteoglycans ( Anal. Biochem. 2007 , 362 , 245 - 251 ; 2007 , 371 , 52 - 61 ). In the present paper, we released O-glycans from some leukemia and epithelial cells using the apparatus, and compared the profiles of O-glycans among these cells after fluorescent labeling of the released glycans with 2-aminobenzoic acid. The fluorescent labeled glycans were analyzed using a combination of HPLC and off-line MALDI-(QIT)TOF mass spectrometry We found that leukemia cells generally showed simple glycan profiles and commonly contained sialyl-T (NeuAcalpha2-3Galbeta1-3GalNAc) and disialyl-T (NeuAcalpha2-3Galbeta1-3(NeuAcalpha2-6)GalNAc) antigens as major O-glycans. In contrast, epithelial cancer cell lines usually showed extremely complex profiles. We found that polylactosamine-type O-glycans were abundantly present in MKN45 cells. Especially, we found characteristic glycans, of which Galbeta1-3 residue of core1 structure is modified with biantennary polylactosamine units. In contrast, this cell line did not contain polylactosamine-type N-glycans ( J. Proteome Res. 2006 , 5 , 88 - 97 ). These results suggest that the different biosynthetic pathways for N- and O-glycans are proposed. The method presented here will accelerate the speed for comprehensive analysis of O-glycans in biological samples and will be a powerful tool for clinical/biochemical analysis in cancer biology.