The transcription factor NF-kappaB regulates a wide variety of genes involved in multiple processes. Although the apparent consensus sequence of DNA binding sites for NF-kappaB (kappaB sites) is very broad, the sites active in any one gene show remarkable evolutionary stability. Using a lentivirus-based methodology for implantation of gene regulatory sequences we show that for genes with two kappaB sites, both are required for activity. Swapping sites between kappaB-dependent genes altered NF-kappaB dimer specificity of the promoters and revealed that two kappaB sites can function together as a module to regulate gene activation. Further, although the sequence of the kappaB site is important for determining kappaB family member specificity, rather than determining the ability of a particular dimer to bind effectively, the sequence affects which coactivators will form productive interactions with the bound NF-kappaB dimer. This suggests that binding sites may impart a specific configuration to bound transcription factors.