The NF-kappa B transcription factors display a high degree of sequence conservation in a domain initially described in the rel oncogene. Two family members, NF-kappa B1 and NF-kappa B2, have distinct DNA binding properties and functionally distinct effects on different enhancers. NF-kappa B1, for example, binds to the kappa B site from the human immunodeficiency virus (HIV) with approximately 15-fold higher affinity than NF-kappa B2. In this study, we have defined regions within the Rel domain which determine DNA binding specificity and interaction with other proteins. We find that the COOH-terminal putative Rel dimerization domain of NF-kappa B1 is required for preferential binding to the HIV kappa B site. In contrast, preferential stimulation of the HIV enhancer by NF-kappa B2 with RelA(p65) is determined by both the NH2- and COOH-terminal Rel domains of NF-kappa B2. These two regions of NF-kappa B2 also mediate preferential synergy with Bcl3. These data suggest that a specific subdomain of the Rel conserved region has evolved to control the fine specificity of DNA binding, and two distinct subregions within the Rel domain determine the specificity of interaction with other transcription factors. These specific Rel-conserved domains therefore determine the specificity of NF-kappa B interactions and contribute to selective gene activation.