The p53 tumor suppressor is phosphorylated in response to various cellular stress signals, such as DNA damage, leading to its release from MDM2 and consequent stabilization and activation as a transcription factor. In human U2OS cells, treatment with adriamycin causes p53 to be phosphorylated on all six serine residues tested, leading to the dissociation of p53 from MDM2 and transcription of the p21 and mdm2 genes. In contrast, in these cells, IPTG-dependent induction of p14ARF, which sequesters MDM2 away from p53, does not lead to detectable phosphorylation of any of the five N-terminal serine residues tested (6, 9, 15, 20, 37). Only C-terminal serine 392 is phosphorylated. However, the increase of p21 and mdm2 mRNAs was indistinguishable following treatment with adriamycin or induction of p14ARF. By using cDNA arrays to examine global p53-dependent gene expression in response to adriamycin or p14ARF, we found that most genes were regulated similarly by the two treatments. However, a subset of p53-regulated genes whose products have proliferative roles or regulate VEGF activity, newly described here, are repressed by p14ARF much more than by adriamycin. We conclude that the phosphorylation of p53 on N-terminal serine residues is not required for increased transcription of the great majority of p53-responsive genes and that the induction of p53 by p14ARF, with little phosphorylation, leads to substantial repression of genes whose products have roles in proliferation.