Benzo[c]chrysene (BcC), an environmental pollutant, is a unique polycyclic aromatic hydrocarbon that possesses both a bay region and a fjord region in the same molecule. We previously demonstrated that both bay region and fjord region terminal rings are involved in the in vitro metabolism of BcC. In the present investigation, we prepared [14-(3)H]BcC and tested the hypothesis that BcC can be activated to both bay region and fjord region diol epoxides in female CD rats. At 6 weeks of age, rats were gavaged with a single dose of [14-(3)H]BcC (5 mg/rat; specific activity, 6.7 Ci/mmol) in 0.5 mL of trioctanoin. During the first 48 h, 20.3% of the dose was eliminated in the feces and 2.8% was eliminated in the urine. After 1 week, cumulatively, 23.2 and 3.5%, respectively, were eliminated. 3-Hydroxybenzo[c]chrysene, 10-hydroxybenzo[c]chrysene, and trans-7,8-dihydroxy-7,8-dihydrobenzo[c]chrysene were the major fecal metabolites. In urine, trans-1,2-dihydroxy-1,2-dihydrobenzo[c]chrysene, 2-hydroxybenzo[c]chrysene, (+/-)-1,t-2,t-3,c-4-tetrahydroxy-1,2,3,4-tetrahydrobenzo[c]chrysene, and (+/-)-9,t-10,t-11,c-12-tetrahydroxy-9,10,11,12-tetrahydrobenzo[c]chrysene were detected, primarily as glucuronic acid and sulfate conjugates. The identification of the two tetraols clearly indicates that both bay region and fjord region diol epoxides are formed as intermediates in the metabolism of BcC in vivo. The second goal of this study was to test the hypothesis that the location of the epoxide moiety (fjord vs bay region) determines the carcinogenic activity. Thus, we compared the carcinogenicity of the bay region (+/-)-anti-1,2-dihydroxy-3,4-epoxy-1,2,3,4-tetrahydrobenzo[c]chrysene and the fjord region (+/-)-anti-9,10-dihydroxy-11,12-epoxy-9,10,11,12-tetrahydrobenzo[c]chrysene in the rat mammary gland. The results clearly showed that the fjord region diol epoxide is a potent mammary carcinogen, while the bay region diol epoxide lacks activity in this model assay. This is the first report on a comparison of mammary cancer induction by fjord and bay region diol epoxides derived from the same molecule. It further supports previous observations that fjord region diol epoxides are more carcinogenic than structurally related bay region diol epoxides.