We previously have shown that polyamine depletion delays apoptosis in rat intestinal epithelial (IEC-6) cells (Am J Physiol Cell Physiol. 278: C480-489, 2000). Here we demonstrate that polyamine depletion inhibits -irradiation induced apoptosis in vitro and in vivo. Pretreatment of IEC-6 cells with 5 mM -difluoromethylornithine (DFMO) for 4 days significantly reduced radiation-induced caspase-3 activity and DNA fragmentation. This protective effect was prevented by the addition of 10 µM exogenous putrescine. Radiation exposure to mice resulted in a high frequency of apoptosis over cells positioned 4^th to 7^th in crypt-villus units. Pretreatment of mice with 2% DFMO in drinking water significantly reduced apoptotic cells from approximately 2.75 to 1.61 per crypt-villus unit, accompanied by significant decreases in caspase-3 levels. Further examination showed that DFMO pretreatment inhibited the radiation-induced increase in the pro-apoptotic protein Bax. Moreover, DFMO pretreatment significantly enhanced the intestinal crypt survival rate by 2.1 fold subsequent to radiation and ameliorated mucosal structural damage. We conclude that polyamine depletion by DFMO inhibits -irradiation induced apoptosis of intestinal epithelia cells both in vitro & in vivo through inhibition of Bax and caspase-3 activity, which leads to attenuation of radiation inflicted intestinal injury. These data indicate that DFMO may be therapeutically useful to counteract the gastrointestinal toxicity caused by chemo-radiotherapy. This is the first demonstration that polyamines are required for apoptosis in vivo.