The nuclear function of the heterodimeric NF-κB transcription factor is regulated in part through reversible acetylation of its RelA subunit. We now demonstrate that the p300 and CBP acetyltransferases play a major role in the in vivo acetylation of RelA, principally targeting lysines 218, 221 and 310 for modification. Analysis of the functional properties of hypoacetylated RelA mutants containing lysine-to-arginine substitutions at these sites and of wild-type RelA co-expressed in the presence of a dominantly interfering mutant of p300 reveals that acetylation at lysine 221 in RelA enhances DNA binding and impairs assembly with IκBα. Conversely, acetylation of lysine 310 is required for full transcriptional activity of RelA in the absence of effects on DNA binding and IκBα assembly. Together, these findings highlight how site-specific acetylation of RelA differentially regulates distinct biological activities of the NF-κB transcription factor complex.