The consequence of mono-(2-ethylhexyl) phthalate (MEHP)-induced injury of testicular Sertoli cells is the Fas-dependent apoptotic elimination of germ cells. In addition to the well-known ability of p53 to regulate the transcription of various apoptosis-associated proteins, p53 also has been implicated in mediating the localization of Fas to the plasma membrane of various cell types in a transcription-independent manner. To resolve the role of p53 in MEHP-mediated testicular toxicity, we used wild-type (p53 ^/ ) and p53 knockout (p53^−/−) mice. A significantly lower incidence of TUNEL-positive germ cells was observed in p53^−/− mice compared to p53 ^/ mice at 1, 1.5, and 24 h after MEHP exposure. In these same mice, an induction of Fas and death receptor-5 (DR5) in testicular membrane preparations was observed only in p53 ^/ mice. Analyses of mRNA levels in testes of p53 ^/ and p53^−/− mice by reverse transcription-polymerase chain reaction revealed that increases in membrane levels of Fas occurred in the absence of their transcriptional up-regulation. Processing of procaspase-8 was observed only in MEHP-treated p53 ^/ mice, and this correlated with the observed incidence of germ cell apoptosis. Interestingly, the p53 status of mice also influenced the stability of c-FLIP (L), a caspase-8 inhibitory protein, that was measured at levels approximately two- to fivefold higher in p53^−/− mice after MEHP-exposure compared to those in p53 ^/ mice. Taken together, these data suggest that MEHP-induced germ cell apoptosis is dependent, in part, on the p53 protein and on its abilities to increase the localization of Fas and DR5 on the germ cell membrane as well as to decrease the cellular levels of c-FLIP (L).