We have provided evidence for a critical role of polyamines in the growth of the hormone-responsive N-nitrosomethyl-urea (NMU)-induced rat mammary tumor in vitro. The present experiments were designed to test whether polyamines are involved in the growth of this experimental tumor in vivo. To test this hypothesis, groups of rats bearing NMU-induced mammary cancers were randomly allocated to receive no treatment or escalating doses of the polyamine biosynthesis inhibitor alpha-difluoromethyl-ornithine (DFMO) (0.5%, 1%, 2%, 3% in drinking water). DFMO inhibited tumor growth in a dose-dependent fashion and consistently reduced tumor putrescine level. To evaluate the time dependency of this effect, additional groups of rats received either no treatment or 2% DFMO for 3, 7, 14, and 21 days. At all times DFMO suppressed tumor putrescine level as well as spermidine to spermine ratio. Finally, exogenous administration of putrescine (200 mg/kg/i.p./day x 21 days) given concomitantly with DFMO restored tumor growth, partially repleted tumor putrescine level, and raised the spermidine to spermine ratio to control levels. Putrescine, given alone, had no significant effect on either tumor polyamine levels or tumor growth. Except for modest weight loss, no major toxicity was encountered. These results indicate that polyamines play an important role in the growth of the NMU rat mammary tumor in vivo. The interaction between polyamines and hormones in supporting NMU mammary tumor growth in vivo remains to be elucidated.