Interferon regulatory factor (IRF)-3 is a critical transcription factor regulating innate immune responses against viral and bacterial infections. Signals activated by various pathogens are integrated by IRF-3 kinase, resulting in the specific phosphorylation of IRF-3 in the cytoplasm. This phosphorylation induces dimerization and association with the coactivators CREB-binding protein/p300, and the resultant complex activates the target genes in the nucleus. However, the phosphorylation sites that determine the active/inactive status of IRF-3 have been a source of controversy. In this study, we generated an antibody that specifically detects the phosphorylation of Ser-386 and used it as a probe. We found: 1) viral infection specifically induces phosphorylation of the Ser-386; 2) recently identified IRF-3 kinases (IKK-i/epsilon and TBK-1) phosphorylate Ser-386 and induce its dimerization; 3) phosphorylation of Ser-386 is exclusively observed with the dimer; 4) mutation at Ser-386 abolishes the dimerization potential; 5) a constitutively active 5D mutant designed to mimic phosphorylation of Ser/Thr residues other than Ser-385 and -386 is secondarily phosphorylated at Ser-386, presumably by an irrelevant kinase. These results strongly suggest that Ser-386 is the target of the IRF-3 kinase and critical determinant for the activation of IRF-3.