Although Neisseria meningitidis can use haemoglobin as an iron source in vitro, the mechanism of haemoglobin-iron uptake is unknown. Using a biotinylated human haemoglobin probe in a solid-phase dot-binding assay, haemoglobin-binding activity was detected in total membranes derived from meningococci grown under iron-limited but not iron-sufficient conditions. In competition binding experiments, bovine and human haemoglobin could abrogate binding. In contrast, no binding inhibition was seen with ferric nitrate, protoporphyrin IX, and iron-loaded human transferrin. The ability of both haemin and catalase, a nonhaemoglobin haem-containing compound, to inhibit binding competitively suggested that the ligand recognized by the binding protein is the haem moiety. Scatchard plot analysis revealed a heterogeneous receptor population. Limited proteolysis with proteinase K abolished binding activity, suggesting a haemoglobin-protein interaction. Detection of activity in a whole-cell binding assay demonstrated that this haemin-binding protein was surface exposed. In a limited survey of meningococcal strains, the presence of haemoglobin-binding activity in all isolates indicated that expression of this binding protein is not serogroup specific.