The existence of an ecto-sialyltransferase (ecto-ST) on B lymphocytes with increasing activity at late maturation stages is shown using a novel flow cytometric enzyme assay. This ecto-ST is effective in reconstituting different surface glycoconjugates on desialylated B cells in the presence of exogenous CMP-NeuAc. We found that this ecto- ST is distinct in its activity from soluble ST released into the culture supernatant. Surface sialylation was independent of the amount of ST secreted into the culture supernatant and followed different kinetics than sialylation of exogenous substrate by soluble ST. Four human B-cell lines representing different maturation stages were analyzed for secreted and ecto-ST activity. The myeloma cell line U266 and the lymphoblastoid cell line JOK-1 showed higher activity of both ST forms than the acute lymphoblastic leukemia B-cell line Nalm-6. ST activity in culture supernatants of U266, JOK-1, and Nalm-6 cells consisted predominantly of the alpha 2,6 ST type with specificity for N- linked oligosaccharides. As an exception, the myeloma cell line IM-9, deficient of alpha 2,6 ST activity, secreted only small amounts of ST and showed low activity of ecto-ST. Sialylation of surface-expressed glycoconjugates by ecto-ST was measured by incubating B-cell lines in the presence of fluorescent CMP-sialic acid. Surface structures labeled with fluorescent sialic acid under this condition were visualized by confocal laser scanning microscopy and fluorescent label was quantitatively assessed by flow cytometric analysis on live cells. Incubation of cells in acidified culture medium, to release possibly receptor-bound ST, did not alter the intensity of cell surface sialylation. Inhibition of internalization and membrane traffic by various approaches (reduced incubation temperature and chloroquine or brefeldin A treatment) did not block surface sialylation. Together, these observations point to cell surface sialylation in B lymphocytes mediated by a cell surface-expressed ecto-ST distinct from the secreted ST form. On desialylated JOK-1 cells, ecto-ST in the presence of exogenous CMP-NeuAc was able to resialylate the B-cell surface sialoglycans CDw75 and HB-6 and major surface glycoproteins of B cells, such as HLA class I and II antigens, transferrin receptor, and surface IgM. In contrast, cell surface glycans of coincubated desialylated erythrocytes were not sialylated by the B-cell ecto-ST. Ecto-alpha 2,6 ST of B cells may be involved in the sialylation of distinct differentiation glycan antigens.