Nucleolin is an ubiquitous nucleolar phosphoprotein involved in fundamental aspects of transcription regulation, cell proliferation and growth. It has also been described as a shuttling molecule between nucleus, cytosol and the cell surface. Several studies have demonstrated that surface nucleolin serves as a receptor for various extracellular ligands implicated in cell proliferation, differentiation, adhesion, mitogenesis and angiogenesis. Previously, we reported that nucleolin in the extranuclear cell compartment is a glycoprotein containing N- and O-glycans. In the present study, we show that glycosylation is an essential requirement for surface nucleolin expression, since it is prevented when cells are cultured in the presence of tunicamycin, an inhibitor of N-glycosylation. Accordingly, surface but not nuclear nucleolin is radioactively labeled upon metabolic labeling of cells with [(3)H]glucosamine. Besides its well-demonstrated role in the internalization of specific ligands, here we show that ligand binding to surface nucleolin could also induce Ca(2+) entry into cells. Indeed, by flow cytometry, microscopy and patch-clamp experiments, we show that the HB-19 pseudopeptide, which binds specifically surface nucleolin, triggers rapid and intense membrane Ca(2+) fluxes in various types of cells. The use of several drugs then indicated that Store-Operated Ca(2+) Entry (SOCE)-like channels are involved in the generation of these fluxes. Taken together, our findings suggest that binding of an extracellular ligand to surface nucleolin could be involved in the activation of signaling pathways by promoting Ca(2+) entry into cells.