Given the fact that increased dietary intake of polycyclic aromatic hydrocarbons (PAHs; a family of environmental toxicants) leads to the formation and development of colon tumors, the ability of the gastrointestinal tract to process these compounds is important from the viewpoint of toxicity/carcinogenesis. Benzo(a)pyrene (BaP), a prototypical PAH compound is released into the environment from automobile exhausts, cigarette smoke, and industrial emissions. Additionally, considerable intake of BaP is expected in people who consume barbecued foods and a diet rich in saturated fat. In exposed animals, BaP becomes activated to potent metabolites that interfere with target organ function and as a consequence cause toxicity and cancer. Therefore, knowledge of BaP metabolism in the digestive system will be of importance in the management of cancers of the digestive tract. The objective of our study was to study the metabolism of BaP by subcellular fractions (nuclear, cytosolic, mitochondrial, and microsomal) of the gastrointestinal tract and liver. Subcellular fractions were isolated by differential centrifugation from the stomach, jejunum, colon, and liver tissues of Apc(Min) mice that received a subchronic dose of 25 μg/kg BaP. The fractions were incubated with 1 and 3 μM BaP. Subsequent to incubation, samples were extracted with ethyl acetate and analyzed for BaP metabolites by reverse-phase HPLC equipped with fluorescence detection. Among the different fractions tested, microsomal BaP metabolism was higher than the rest of the fractions in all the samples analyzed. Additionally, a BaP exposure concentration-dependent effect on metabolite levels generated by the subcellular fractions was recorded. The BaP metabolites identified were the following: BaP-9,10-diol; BaP-4,5-diol; BaP-7,8-diol; 9(OH) BaP; 3(OH) BaP; BaP-3,6-dione; and BaP-6,12-dione. While the diol group of metabolites was frequently detected, among diones, the 3,6 and 6,12-dione metabolites were infrequently detected. Among the diol metabolites, the preponderance of BaP-7,8-dihydrodiol is interesting, since this metabolite is a precursor to the DNA-reactive BaP-7,8-dihydrodiol epoxide (BPDE) that has been linked to BaP-induced cancer.