Lactoferrin (Lf), an iron-sequestering glycoprotein, predominates in mucosal secretions, where the level of free extracellular iron (10⁻¹⁸ M) is not sufficient for bacterial growth. This represents a mechanism of resistance to bacterial infections by prevention of colonization of the host by pathogens. In this study we were able to show that Streptococcus pneumoniaespecifically recognizes and binds the iron carrier protein human Lf (hLf). Pretreatment of pneumococci with proteases reduced hLf binding significantly, indicating that the hLf receptor is proteinaceous. Binding assays performed with 63 clinical isolates belonging to different serotypes showed that 88% of the tested isolates interacted with hLf. Scatchard analysis showed the existence of two hLf-binding proteins with dissociation constants of 5.7 × 10⁻⁸and 2.74 × 10⁻⁷ M. The receptors were purified by affinity chromatography, and internal sequence analysis revealed that one of the S. pneumoniae proteins was homologous to pneumococcal surface protein A (PspA). The function of PspA as an hLf-binding protein was confirmed by the ability of purified PspA to bind hLf and to competitively inhibit hLf binding to pneumococci.S. pneumoniae may use the hLf-PspA interaction to overcome the iron limitation at mucosal surfaces, and this might represent a potential virulence mechanism.