It is well established that both MDM2 and MDMX are essential factors for maintaning tumor suppressor p53 in its latent form. We have previously shown that MDMX proteins were rapidly degraded by MDM2 in response to DNA damage, a phenomenon which was a critical event in optimal p53 activation. Recent mouse genetic studies have revealed that the expression level of MDMX critically influences the survival of mice after whole-body ionizing radiation exposure. Many in vivo and in vitro studies have shown that MDM2 functions as a E3 ubiquitin ligase to ubiquitinate and down-regulate P53 protein, and forms a negative feedback loop with. However, it is still controversial whether MDMX has a functional activity in the control of P53 stability. Although our studies suggested that MDMX forms heterodimer with MDM2 and enhances MDM2 E3 ubiquitin ligase activity in cells, the underlying molecular mechanisms of how MDMX regulates p53 are still unclear. In order to investigate further possible molecular mechanisms of the p53 regulation by MDM2 and MDMX, we are trying to develop an in vitro assay system in which ubiquitination studies of p53, MDM2 and MDMX can be performed. Here, we present data validating this assay system and report a novel mechanism by which the MDM2/MDMX heterocomplex regulates the p53 ubiquitination and activation in response to DNA damage.