We examined the interactions of live and lysed spirochetes with innate immune cells. THP-1 monocytoid cells were activated to comparable extents by live Borrelia burgdorferi and by B. burgdorferi and Treponema pallidum lysates but were poorly activated by live T. pallidum. Because THP-1 cells poorly internalized live spirochetes, we turned to an ex vivo peripheral blood mononuclear cell system that would more closely reflect spirochete-mononuclear phagocyte interactions that occur during actual infection. In this system, B. burgdorferi induced significantly greater monocyte activation and inflammatory cytokine production than did borrelial lysates or T. pallidum, and only B. burgdorferi elicited gamma interferon (IFN-γ) from NK cells. B. burgdorferi was phagocytosed avidly by monocytes, while T. pallidum was not, suggesting that the enhanced response to live B. burgdorferi was due to phagocytosis of the organism. When cytochalasin D was used to block phagocytosis of live B. burgdorferi, cytokine production decreased to levels comparable to those induced by B. burgdorferi lysates, while the IFN-γ response was abrogated altogether. In the presence of human syphilitic serum, T. pallidum was efficiently internalized and initiated responses resembling those observed with live B. burgdorferi, including the production of IFN-γ by NK cells. Depletion of monocytes revealed that they were the primary source of inflammatory cytokines, while dendritic cells (DCs) directed IFN-γ production from innate lymphocytes. Thus, phagocytosis of live spirochetes initiates cell activation programs in monocytes and DCs that differ qualitatively and quantitatively from those induced at the cell surface by lipoprotein-enriched lysates. The greater stimulatory capacity of B. burgdorferi versus T. pallidum appears to be explained by the successful recognition and phagocytosis of B. burgdorferi by host cells and the ability of T. pallidum to avoid detection and uptake by virtue of its denuded outer membrane rather than by differences in surface lipoprotein expression.