Bovine lactoferrin (bLf) is known to damage the outer membrane of Gram-negative bacteria by binding to bacterial lipopolysaccharide (LPS). We report that LPS is released from bacterial outer membranes also when apo- or metal-saturated Lf is separated from bacterial cells by a dialysis membrane. This process occurs in phosphate-buffered saline with no added Ca²⁺and Mg²⁺and is hindered by addition of these cations. The effect of bLf is similar to that induced by EDTA and has been ascribed to chelation of Ca²⁺. In fact, it may be envisaged that Ca²⁺-binding sites on LPS have different affinities and that bLf can remove those ions that are more weakly bound. Ca²⁺binding does not alter Lf iron-binding properties significantly or its UV and CD spectral features but brings about changes in the FT-IR bands due to carboxylate residues. Ca²⁺binding is characterized by an apparent dissociation constant of 6 µM and a stoichiometry of 1.55 Ca²⁺per Lf molecule; it enhances bLf stability towards chemical and thermal denaturation. The increase in stability takes place in both the apo- and iron-saturated forms but not in the desialilated protein, indicating that the carboxylate groups of the sialic acid residues present on two of the glycan chains are involved in Ca²⁺binding.Key words: lactoferrin, calcium, antibacterial activity, lipopolysaccharides, protein stability, denaturation.