T-lymphocytic leukemia expresses complex, branched O-linked oligosaccharides on a major sialoglycoprotein, leukosialin. Leukocytes express a major sialoglycoprotein, leukosialin, of which the apparent molecular weight (mol wt) can be variable according to the differences in O-glycans attached to this molecule. In the present study, we analyzed the structures of O-glycans attached to leukosialin present in various T-lymphocytic leukemia cells. T-lymphoid cells from patients with acute T-lymphocytic leukemia express a large amount of the branched hexasaccharides, NeuNAc alpha 2----3Gal beta 1----3(NeuNAc alpha 2----3Gal beta 1----4GlcNAc beta 1----6)GalNAc, which are also expressed in activated normal T lymphocytes, but that are almost absent in resting normal T lymphocytes. T-lymphoid cells from patients with chronic T-lymphocytic leukemia, on the other hand, mainly express the tetrasaccharides NeuNAc alpha 2----3Gal beta 1----3(NeuNAc alpha 2----6)GalNAc on leukosialin, but they also express a small significant amount of the hexasaccharides. The same hexasaccharides can be detected in thymocytes. The increased amount of the hexasaccharides in acute leukemia is associated with increased activity of beta 1----6 GlcNAc-transferase, a key enzyme in forming the hexasaccharides. Immunoblot analysis of cell lysates showed that monoclonal antibody (MoAb) T-305 reacts preferentially with leukosialin of high mol wt containing the hexasaccharides. These findings indicate that T-lymphocytic leukemia cells reexpress the oligosaccharides present in immature cells. 