Serum pools were collected from rabbits bled at various times after intra-testicular infection with Treponema pallidum ssp. pallidum. These were tested for their ability to opsonize T. pallidum and promote killing of the organisms by macrophages. Compared to normal sera, significant opsonization was first seen on day 10 of infection as measured by both ingestion (P < 0.001) and macrophage-mediated killing (P = 0.006); significant levels of functional antibodies persisted through 300 days of infection. Although opsonic activity peaked early in infection, antibodies that promoted optimal macrophage-mediated killing developed much later, suggesting that these two functions may represent activities of antibodies with differing specificities or affinities. The initial development of antibodies that augment both phagocytosis and killing corresponds with the in vivo clearance of treponemes from the primary site of infection. These observations support the hypothesis that macrophages are the major effector mechanism for elimination of T. pallidum during early syphilis infection.