Inhibitory effects of alpha-difluoromethylornithine (DFMO) on urinary bladder carcinogenesis were examined using the heterotopically transplanted rat urinary bladder (HTB) model. Male Fischer rats with an HTB were arbitrarily divided into four groups. Group 1 rats received into the HTBs 0.25 mg of N-methyl-N-nitrosourea (MNU) once a week for 3 weeks, followed by instillation twice a week of 0.5 ml of 2% DFMO dissolved in normal rat urine. Group 2 rats received the same amount of MNU, followed by instillation of urine without DFMO. Group 3 rats received a single dose of 0.25 mg of MNU, followed by instillation twice a week of urine containing 2% DFMO. Group 4 rats were treated as those in Group 3 but without DFMO. At 8, 14, and 20 weeks after the last MNU administration, urothelial polyamine levels and [3H] thymidine incorporation by the urothelium of HTBs were determined in nine rats of Groups 1 and 2. The remaining animals of Groups 1 and 2 were killed 25 weeks after the beginning of MNU injection, while those of Groups 3 and 4, 30 weeks after the MNU treatment. The contents of 3 polyamines (putrescine, spermidine, and spermine) in urothelial cells were significantly lower in Group 1 as compared with Group 2. The incidences of carcinoma were significantly lower in the groups treated with DFMO (p less than 0.001, Group 1 versus Group 2; p less than 0.005, Group 3 versus Group 4). These observations indicate that administration of DFMO inhibits (or retards) bladder carcinogenesis in HTBs. A possible mechanism for this effect is suppression of polyamine biosynthesis and proliferation of bladder epithelial cells.