p300 and CREB-binding protein (CBP) act as multifunctional regulators of p53 via acetylase and polyubiquitin ligase (E4) activities. Prior work in vitro has shown that the N-terminal 595 aa of p300 encode both generic ubiquitin ligase (E3) and p53-directed E4 functions. Analysis of p300 or CBP-deficient cells revealed that both coactivators were required for endogenous p53 polyubiquitination and the normally rapid turnover of p53 in unstressed cells. Unexpectedly, p300/CBP ubiquitin ligase activities were absent in nuclear extracts and exclusively cytoplasmic. Consistent with the cytoplasmic localization of its E3/E4 activity, CBP deficiency specifically stabilized cytoplasmic, but not nuclear p53. The N-terminal 616 aa of CBP, which includes the conserved Zn2+-binding C/H1-TAZ1 domain, was the minimal domain sufficient to destabilize p53 in vivo, and it included within an intrinsic E3 autoubiquitination activity and, in a two-step E4 assay, exhibited robust E4 activity for p53. Cytoplasmic compartmentalization of p300/CBP's ubiquitination function reconciles seemingly opposed functions and explains how a futile cycle is avoided—cytoplasmic p300/CBP E4 activities ubiquitinate and destabilize p53, while physically separate nuclear p300/CBP activities, such as p53 acetylation, activate p53.