To evaluate the role of polyamines in mediating the effect of estrogen on growth of the rat uterus, we examined the effect of an irreversible inhibitor of ornithine decarboxylase (ODC), 5 alpha-difluoromethylornithine (DFMO), on estrogen-stimulated uterine growth. Estradiol alone evoked two major surges of ODC activity at 5 h and at 16 h, and 2-fold increases in the concentration of spermidine in the uterus. When DFMO was administered to either intact or ovariectomized immature (20 day-old) rats for 2 or 6 days prior to and then during the 1 or 3 days of estradiol treatment, uterine ODC activity was completely suppressed as was the associated increase in spermidine concentration, but the magnitude of uterine growth stimulated by estradiol was equally great in the presence or absence of DFMO. These observations suggest that although uterine growth stimulation by estrogen is normally accompanied by marked increases in ODC activity and spermidine accumulation, these changes are not essential for hormonally regulated uterine cell proliferation in vivo.