WR1065 is an aminothiol with selective cytoprotective effects in normal compared to cancer cells, which is used to protect tissues against the damaging effect of radiation and chemotherapeutic drugs. WR1065 has been shown to induce wild-type p53 accumulation and activation in cultured cells, suggesting a role of p53 in cytoprotection. However, the molecular mechanisms by which WR1065 activates p53 remain unclear. Here, we demonstrated that p53 accumulation by WR1065 in MCF-7 cells did not result from the formation of DNA-damage as measured by DNA fragmentation and Comet assay, nor from oxidative stress as detected by measurement of glutathione levels, lipid peroxidation and reactive oxygen species production. p53 activation by WR1065 was not prevented by inhibition of PI-3 kinases, and was still detectable in MCF-7 cells stably transfected with the oncoprotein E6, which repressed p53 induction by DNA damage. These data provided evidence that WR1065 induces p53 by a pathway different than the one elicited by DNA-damage. Direct reduction by WR1065 of key cysteines in p53 may play an important role in this alternative pathway, as shown by the fact that WR1065 activated the redox-dependent, DNA-binding activity of p53 in vitro.