Lactoferrin (Lf), a multifunctional molecule present in mammalian secretions and blood, plays important roles in host defense and cancer. Indeed, Lf has been reported to inhibit the proliferation of cancerous mammary gland epithelial cells and manifest a potent antiviral activity against human immunodeficiency virus and human cytomegalovirus. The Lf-binding sites on the cell surface appear to be proteoglycans and other as yet undefined protein(s). Here, we isolated a Lf-binding 105 kDa molecular mass protein from cell extracts and identified it as human nucleolin. Medium-affinity interactions ( approximately 240 nm) between Lf and purified nucleolin were further illustrated by surface plasmon resonance assays. The interaction of Lf with the cell surface-expressed nucleolin was then demonstrated through competitive binding studies between Lf and the anti-human immunodeficiency virus pseudopeptide, HB-19, which binds specifically surface-expressed nucleolin independently of proteoglycans. Interestingly, binding competition studies between HB-19 and various Lf derivatives in proteoglycan-deficient hamster cells suggested that the nucleolin-binding site is located in both the N- and C-terminal lobes of Lf, whereas the basic N-terminal region is dispensable. On intact cells, Lf co-localizes with surface nucleolin and together they become internalized through vesicles of the recycling/degradation pathway by an active process. Morever, a small proportion of Lf appears to translocate in the nucleus of cells. Finally, the observations that endocytosis of Lf is inhibited by the HB-19 pseudopeptide, and the lack of Lf endocytosis in proteoglycan-deficient cells despite Lf binding, point out that both nucleolin and proteoglycans are implicated in the mechanism of Lf endocytosis.