It has been shown previously that sub-complexes of the 26 S proteasome can regulate gene expression via non-proteolytic mechanisms. One such mechanism is the disruption of activator.promoter complexes in an ATP-dependent fashion, which was discovered in the context of the yeast Gal4 system. This activity strongly inhibits Gal4-driven gene expression unless the activator is mono-ubiquitylated, which protects it from the ATPases. To address whether this paradigm is also applicable to medically important mammalian transcriptional activators we report here a study of the interaction of the proteasomal ATPases with p53. It is shown that p53 binds directly to the ATPases via its C-terminal tetramerization and regulatory domain and that p53.promoter complexes are indeed vulnerable to ATPase-dependent disruption by the ATPase complex in vitro. Knockdown of one of the ATPases, Rpt6, in living cells results in increased occupancy of the p21(waf1) promoter by p53 and increased expression of the gene, consistent with the idea that the proteasomal ATPases negatively regulate p53 function in a non-proteolytic fashion.