P53 is inactivated in tumors by mechanisms other than mutations in the p53 gene itself. To gain insight into the mechanisms by which this inactivation occurs, we chemically mutagenized A1-5 cells expressing high levels of temperature sensitive p53 val135 (tsp53) and selected for clones that were capable of growth at the permissive temperature for p53 activation. We expanded 22 clones (ALTR cells for A1-5 Low Temperature Resistant) that could grow at the permissive temperature. Most exhibited cytoplasmic sequestration as the mechanism by which p53 was inactivated. We show here that this cytoplasmically sequestered tsp53 protein is maintained in a mutant conformation. Only in clones with nuclear localized p53 is it also expressed in the wild-type conformation suggesting that subcellular localization of tsp53 is important in determining the conformation of the protein. Consistent with this, we show that the changes in conformation of p53 in A1-5 and SK-N-SH cells induced by ionizing radiation also correlate with nuclear translocation of p53. We suggest that nuclear translocation of p53 can result in a change in the conformation from mutant to wild-type but that these may be two separable events. Oncogene (2000) 19, 4042 ± 4049.