The nuclear function of the NF-κB transcription factor is regulated by reversible acetylation of its RelA subunit. The study demonstrates that p300 and CBP acetyltransferases play a major role in the in vivo acetylation of RelA, primarily targeting lysines 218, 221, and 310. Analysis of hypoacetylated RelA mutants and wild-type RelA co-expressed with a dominant interfering mutant of p300 reveals that 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 without affecting DNA binding or IκBα assembly. These findings highlight how site-specific acetylation of RelA differentially regulates distinct biological activities of the NF-κB transcription factor complex.The nuclear function of the NF-κB transcription factor is regulated by reversible acetylation of its RelA subunit. The study demonstrates that p300 and CBP acetyltransferases play a major role in the in vivo acetylation of RelA, primarily targeting lysines 218, 221, and 310. Analysis of hypoacetylated RelA mutants and wild-type RelA co-expressed with a dominant interfering mutant of p300 reveals that 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 without affecting DNA binding or IκBα assembly. These findings highlight how site-specific acetylation of RelA differentially regulates distinct biological activities of the NF-κB transcription factor complex.