Differences in susceptibility to N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN)-induced urinary bladder carcinogenesis between two substrains of male Sprague-Dawley rats were examined. One substrain was SD/gShi, which has spontaneous hypospermatogenesis, and the other was SD/cShi, which is a sister strain of SD/gShi, and has normal testis but spontaneous hydronephrosis. SD/gShi rats had a lower incidence of urinary bladder tumors and had lower 5-bromo-2'-deoxyuridine labeling indices in the urinary bladder epithelium than SD/cShi rats when BBN was given. SD/gShi rats had significantly lower urinary concentrations of N-butyl-N-(3-carboxypropyl)nitrosamine (BCPN), which is a metabolite and proximate carcinogen of BBN. In vitro analysis also showed significantly less BCPN formation, using an S9 mix derived from the liver and kidney, in SD/gShi rats than in SD/cShi rats. BCPN formation in vitro was markedly inhibited by non-selective cytochrome P450 (CYP) inhibitors, but not alcohol dehydrogenase inhibitor. However, analysis of CYP proteins including hepatic CYP1A1/2, 2B1/2, 2E1, and 3A2 and renal CYP2E1 and 3A2 revealed no significant variation in levels in either tissue in the groups. There were also no significant intergroup differences in the mutagenicity of carcinogens, including heterocyclic amines and N-nitrosamines, activated by CYP1A1/2 and CYP2E1 and/or CYP2B1/2, respectively. These results suggest that SD/gShi rats are less susceptible to BBN, possibly because less BCPN is produced by CYP isoforms other than those investigated. A contribution of CYP4B1 to the strain difference is also possible.