Signal transducer and activator of transcription 3 (STAT3) and nuclear factor kappaB (NF-kappaB) are important transcription factors involved in glomerulonephritis and other inflammatory processes, including transcription of the inducible nitric oxide synthase (iNOS) gene. The ability of STAT3 to interact physically with NF-kappaB p65 in glomerular mesangial cells and thereby to inhibit NF-kappaB-mediated transactivation of the iNOS gene was demonstrated previously. STAT3 is a modular protein with several structurally and functionally defined domains. For defining STAT3 domains that interact with NF-kappaB p65, (35)S-labeled proteins that corresponded to each STAT3alpha domain were synthesized, and their ability to bind specifically a GST-NF-kappaB p65 fusion protein in GST pulldown assays was tested. The coiled-coil and DNA-binding domains were specifically retained by GST-NF-kappaB p65, whereas the N-terminal, linker domain, Src homology 2 domain, and transcriptional activation domain failed to interact with NF-kappaB p65. Deletion of the region L(358) through I(369) of the STAT3 DNA-binding domain greatly reduced binding to GST-NF-kappaB p65. Alanine substitution mutations at four highly conserved residues-L(358), N(359), K(363), and V(366)-in this region greatly abrogated the ability of STAT3 to bind NF-kappaB p65. Moreover, in contrast to the transrepression afforded by wild-type STAT3alpha, a STAT3alpha construct harboring these mutations, failed to suppress endogenous NO production and to transrepress iNOS promoter-reporter and kappaB element-reporter constructs in IL-beta-stimulated mesangial cells. These data reveal a novel role for the DNA-binding domain in the physical and functional coupling of STAT3 to NF-kappaB p65 that is important for regulating the transcriptional activity of iNOS and likely other NF-kappaB p65 responsive genes that are important for mesangial cell responses.