Subcellular localization guided by IkappaBalpha is crucial for regulation of nuclear factor-kappaB function. Here, we show that p65 Rel homology domain phosphorylation mutants are transported into the nucleus after IkappaBalpha degradation, but as a consequence of lower IkappaBalpha levels their relocation to the cytosol is blocked. We demonstrate that phosphorylation of residues S205, S276, and S281 of p65 is not required for interaction between p65 and IkappaBalpha, but is pivotal for regulating cellular IkappaBalpha levels by positively affecting gene synthesis. Our findings indicate that reduction of phosphorylation leads to nuclear retention of p65, which might be partly responsible for altered transcriptional behavior of p65 serine mutants.