The inhibitory effect of oral administration of alpha-difluoromethylornithine (DFMO) on urinary bladder carcinogenesis induced by N-butyl-N-(4-hydroxybutyl)-nitrosamine (BHBN) was explored. Since DFMO in the drinking water at 0.5% and 0.2% had been demonstrated to inhibit tumorigenesis, lower doses (0.2%, 0.1%, 0.03% and 0.01%) of DFMO were examined in the present study. After six-week treatment with the drinking water containing 0.05% BHBN, water containing DFMO at 0.2%, 0.1%, 0.03%, 0.01% or 0% was administered during the subsequent 34 weeks. Incidence of bladder carcinoma was 15/35 (43%), 14/35 (40%), 21/35 (60%), 20/35 (57%) and 27/35 (77%) in the 0.2%, 0.1%, 0.03%, 0.01% and 0% DFMO groups, respectively. Stastistical analysis indicated significant tumor suppression in the 0.2% and 0.1% DFMO groups. Tumor multiplicity and size were not affected by DFMO treatment. No untoward effects were demonstrated in body weight gain or examination of pertinent organs. Our data indicate that bladder carcinogenesis induced by six-week exposure to 0.05% BHBN is significantly inhibited by daily administration of DFMO at the level of 0.1% or higher in drinking water.