In this study, we sought to investigate the mechanism of the proapoptotic function of Egr-1 in relation to p53 status in normal isogenic cell backgrounds by using primary MEF cells established from homozygous (Egr-1-/-) and heterozygous (Egr-1+/-) Egr-1 knock-out mice. Ionizing radiation caused significantly enhanced apoptosis in Egr-1+/- cells (22.8%; p < 0.0001) when compared with Egr-1-/- cells (3.5%). Radiation elevated p53 protein in Egr-1+/- cells in 3-6 h. However, in Egr-1-/- cells, the p53 protein was down-regulated 1 h after radiation and was completely degraded at the later time points. Radiation elevated the p53-CAT activity in Egr-1+/- cells but not in Egr-1 -/-cells. Interestingly, transient overexpression of EGR-1 in p53-/- MEF cells caused marginal induction of radiation-induced apoptosis when compared with p53+/+ MEF cells. Together, these results indicate that Egr-1 may transregulate p53, and both EGR-1 and p53 functions are essential to mediate radiation-induced apoptosis. Rb, an Egr-1 target gene, forms a trimeric complex with p53 and MDM2 to prevent MDM2-mediated p53 degradation. Low levels of Rb including hypophosphorylated forms were observed in Egr-1-/- MEF cells before and after radiation when compared with the levels observed in Egr-1+/- cells. Elevated amounts of the p53-MDM2 complex and low amounts of Rb-MDM-2 complex were observed in Egr-1-/- cells after radiation. Because of a reduction in Rb binding to MDM2 and an increase in MDM2 binding with p53, p53 is directly degraded by MDM2, and this leads to inactivation of the p53-mediated apoptotic pathway in Egr-1-/- MEF cells. Thus, the proapoptotic function of Egr-1 may involve the mediation of Rb protein that is essential to overcome the antiapoptotic function of MDM2 on p53.