Signaling via the NF-kappaB cascade is critical for innate recognition of microbial products and immunity to infection. As a consequence, this pathway represents a strong selective pressure on infectious agents and many parasitic, bacterial and viral pathogens have evolved ways to subvert NF-kappaB signaling to promote their survival. Although the mechanisms utilized by microorganisms to modulate NF-kappaB signaling are diverse, a common theme is targeting of the steps that lead to IkappaB degradation, a major regulatory checkpoint of this pathway. The data presented here demonstrate that infection of mammalian cells with Toxoplasma gondii results in the activation of IKK and degradation of IkappaB. However, despite initiation of these hallmarks of NF-kappaB signaling, neither nuclear accumulation of NF-kappaB nor NF-kappaB-driven gene expression is observed in infected cells. However, this defect was not due to a parasite-mediated block in nuclear import, as general nuclear import and constitutive nuclear-cytoplasmic shuttling of NF-kappaB remain intact in infected cells. Rather, in T. gondii-infected cells, the termination of NF-kappaB signaling is associated with reduced phosphorylation of p65/RelA, an event involved in the ability of NF-kappaB to translocate to the nucleus and bind DNA. Thus, these studies demonstrate for the first time that the phosphorylation of p65/RelA represents an event downstream of IkappaB degradation that may be targeted by pathogens to subvert NF-kappaB signaling.