Acetylation of RelA at discrete sites regulates distinct nuclear functions of NF-κB. The study shows that p300 and CBP acetyltransferases play a major role in the in vivo acetylation of RelA, primarily targeting lysines 218, 221, and 310. Acetylation at lysine 221 enhances DNA binding and impairs assembly with IκBα, while acetylation at lysine 310 is required for full transcriptional activity of RelA. These findings highlight how site-specific acetylation of RelA differentially regulates distinct biological activities of the NF-κB transcription factor complex. The study also demonstrates that acetylation of RelA at lysine 221 enhances its binding affinity for the κB enhancer and prevents its association with IκBα. Acetylation of lysine 221 and possibly lysine 218 regulates assembly with IκBα. The RelA-KR mutant, which lacks acetylation at these sites, predominantly localizes in the cytoplasm. The study concludes that acetylation of RelA at distinct sites differentially regulates various biological functions of NF-κB. Acetylation of lysine 310 is required for full transactivation by the NF-κB complex, while acetylation of lysine 221 enhances RelA binding to the κB enhancer and impairs assembly with IκBα. Lysines 218 and 221 are highly conserved within all Rel family members, including Dorsal from Drosophila. The possibility that these evolutionarily conserved lysine residues are targets for reversible acetylation and contribute to the regulation of the biological functions of other Rel factors remains an intriguing possibility.Acetylation of RelA at discrete sites regulates distinct nuclear functions of NF-κB. The study shows that p300 and CBP acetyltransferases play a major role in the in vivo acetylation of RelA, primarily targeting lysines 218, 221, and 310. Acetylation at lysine 221 enhances DNA binding and impairs assembly with IκBα, while acetylation at lysine 310 is required for full transcriptional activity of RelA. These findings highlight how site-specific acetylation of RelA differentially regulates distinct biological activities of the NF-κB transcription factor complex. The study also demonstrates that acetylation of RelA at lysine 221 enhances its binding affinity for the κB enhancer and prevents its association with IκBα. Acetylation of lysine 221 and possibly lysine 218 regulates assembly with IκBα. The RelA-KR mutant, which lacks acetylation at these sites, predominantly localizes in the cytoplasm. The study concludes that acetylation of RelA at distinct sites differentially regulates various biological functions of NF-κB. Acetylation of lysine 310 is required for full transactivation by the NF-κB complex, while acetylation of lysine 221 enhances RelA binding to the κB enhancer and impairs assembly with IκBα. Lysines 218 and 221 are highly conserved within all Rel family members, including Dorsal from Drosophila. The possibility that these evolutionarily conserved lysine residues are targets for reversible acetylation and contribute to the regulation of the biological functions of other Rel factors remains an intriguing possibility.