Abstract NF-κB plays a major role in regulating the immune system. Therefore, alterations in NF-κB activity have profound effects on many immunopathologies, including inflammation, autoimmunity, and lymphoid neoplasia. We investigated the effects of estrogen (17β-estradiol) on NF-κB in C57BL/6 mice since estrogen is a natural immunomodulator and we have recently reported that estrogen up-regulates several NF-κB-regulated proteins (inducible NO synthase, IFN-γ, and MCP-1). We found that in vivo estrogen treatment had differential effects on NF-κB family members. Estrogen profoundly blocked the nuclear translocation of p65, c-Rel, and Rel-B, partially blocked p52, but permitted translocation of p50. Despite blockade of both the classical (p65/p50) and alternative (RelB/p52) NF-κB activation pathways, estrogen induced constitutive NF-κB activity and increased the levels of cytokines regulated by NF-κB (IL-1α, IL-1β, IL-10, and IFN-γ). Studies involving a NF-κB inhibitor confirmed a positive regulatory role of NF-κB on these cytokines. Remarkably, estrogen selectively induced B cell lymphoma 3 (Bcl-3), which is known to associate with p50 to confer transactivation capabilities, thereby providing a potential link between observed p50 DNA-binding activity and estrogen up-regulation of NF-κB transcriptional activity. Chromatin immunoprecipitation assays confirmed that Bcl-3 bound to the promoter of the NF-κB-regulated inducible NO synthase gene in cells from estrogen-treated mice. Estrogen appeared to act at the posttranscriptional level to up-regulate Bcl-3 because mRNA levels in splenocytes from placebo- and estrogen-treated mice were comparable. The novel findings of differential regulation of NF-κB proteins by estrogen provide fresh insight into potential mechanisms by which estrogen can regulate NF-κB-dependent immunological events.