Although adenovirus 5 E1A-binding protein (BS69) is a nuclear protein acting as a transcriptional repressor, we found by an yeast two-hybrid and human cell immunoprecipitation another cytoplasmic function for this protein. BS69 bound Toll-interleukin 1 receptor domain (TIR)-containing adaptor molecule-1 (TICAM-1) (also named TRIF), an adaptor protein that couples with TLR3 around the endosome. BS69 translocated from the nucleus to the cytoplasm when cells were stimulated with dsRNA or transfected with TICAM-1. Confocal analysis of cells with over-expressed TICAM-1 or those stimulated with dsRNA revealed the characteristic "TICAM-1 speckle", which reflects signalosome formation necessary for the activation of NF-kappaB and IFN-regulatory factor (IRF)-3. BS69 was involved in the TICAM-1 complex, and the activation of NF-kappaB/IRF-3 followed by cytokine production was augmented in the presence of BS69 overexpression. Knockdown of endogenous BS69 resulted in a decrease of IFN-beta induction, suggesting that BS69 is a positive regulator for the TLR3-TICAM-1 pathway. These results, together with a recent report showing the negative regulatory properties of BS69 in NF-kappaB activation by EBV-derived latent membrane protein 1, suggest that BS69 harbors dual modes of cytoplasmic NF-kappaB regulation, positively in the TICAM-1 pathway and negatively in the latent membrane protein 1 pathway.