Lactoferrin-binding or -associated proteins were identified in Treponema pallidum subspecies pallidum and Treponema denticola by affinity column chromatography using human lactoferrin and detergent-solubilized, radiolabelled spirochaetes. Two discrete polypeptides of T. pallidum with masses of 45 and 40 kDa and a broad band from 29-34 kDa exhibited association with human apo- and partially ferrated lactoferrin. T. denticola produced two proteins that associated with a lactoferrin affinity matrix (50 and 35 kDa). T. pallidum and T. denticola did not associate with soluble, human transferrin in parallel experiments. Soluble human lactoferrin competed with all lactoferrin-associated proteins from T. pallidum and T. denticola in competitive-binding assays. However, the T. denticola proteins dissociated from a lactoferrin-affinity matrix in the presence of differing concentrations of unlabelled, soluble lactoferrin competitor. Treatment with phospholipase D altered migration of the diffuse 29-34 kDa band of T. pallidum suggesting that the polypeptide was lipid-modified. Each of the lactoferrin-binding proteins from T. pallidum and T. denticola reacted with pooled rabbit syphilitic antisera. The lactoferrin-binding proteins of T. pallidum reacted with human sera from patients at all stages of syphilis. In addition, a monoclonal antibody generated against the 45 kDa polypeptide of T. pallidum crossreacted with the 29-34 kDa protein.