The tumor suppressor p53 protein is expressed at low levels under normal conditions. The subcellular localization and functional activation of p53 are influenced by diverse stress signals. p53 in cytoplasm exerts intrinsic 3'→5' exonuclease activity with various RNA and DNA substrates. ssRNAs containing an adenosine and uridine-rich (ARE) element are permissive targets for p53-mediated degradation. The analysis of the exonuclease activity in cytoplasm with activated p53 induced by various drug treatments or following γ-irradiation revealed that the expression of p53 exonuclease activity in response to stress signals is heterogeneous. Various genotoxic and non-genotoxic agents upregulate p53 yet have different effects on expression of exonuclease activity with ARE RNA but not with DNA substrate. Ribonuclease activity is enhanced in cytoplasmic extracts of HCT116 (p53+/+) cells exposed to γ-irradiation or treated by the non-genotoxic drug AS101 but decreased following treatment by genotoxic (e.g., doxorubicin) or non-genotoxic (e.g., DFMO) agents, thus indicating that p53 exonuclease activity is dependent on the specific stress and nature of the substrate. Apparently, the disparity in expression of p53 ribonuclease activity after each treatment is attributable to the different post-treatment response and to two posttranscriptional events: the interaction of RNA-binding HuR protein with ARE RNA protects the substrate from degradation by p53 and/or decrease in p53 ARE RNA binding capacity due to phosphorylation at Ser392 leads to reduction in p5 ribonuclease activity. Our results provide new insights into p53 exonuclease function and into the mechanisms behind the regulation ARE-RNA degradation by p53 under different cellular conditions.