The role of polyamines as mediators of the mitogenic effect of 17 beta-estradiol (E2) was investigated in the N-nitrosomethylurea [(NMU) CAS: 684-93-5; N-methyl-N-nitrosourea]-induced Sprague-Dawley rat mammary tumor grown in the soft agar clonogenic assay under serum-free medium conditions. Inhibition of polyamine biosynthesis with alpha-difluoromethylornithine (DFMO) (1 mM), an irreversible inhibitor of ornithine decarboxylase, completely blocked the growth-promoting effect of E2 (10(-8) M) in this system. Exogenous administration of putrescine (2.5 mM), spermidine (0.1 mM), and spermine (0.1 mM) reversed the inhibitory effect of DFMO and completely restored the action of E2. These polyamines, however, had no effect on tumor growth when they were added alone in identical concentrations. The results indicate that polyamines are essential but probably not sufficient in mediating the E2 effect on the growth of the NMU-induced mammary tumor under these experimental conditions.