We have investigated the mutagenicity of benzo[a]pyrene (B[a]P) in small intestine using the Dlb-1 locus assay in the mouse. Administration of B[a]P by the oral and i.p. routes had markedly different effects on the number of Dlb-1 mutations and the pattern of induction of cytochrome P-4501A1 (CYP1A1). In Ahr-responsive animals i.p. injection resulted in marked induction in crypt cells along the length of the small intestine, with some induction in the villus cells. In contrast, after oral administration, CYP1A1 induction was evident only in the villus cells, and this declined distally. The intensity and speed of induction in Ahr-responsive animals was such that the genotoxic effect of a single injection of B[a]P could not be augmented by prior treatment with non-genotoxic inducers such as β-napthoflavone and TCDD. Oral B[a]P treatment resulted in a decrease in the number of mutations when compared with the i.p. route. Studies in congenic Ahr-non-responsive versus Ahr-responsive mice indicated that induction of CYP1A1 was associated with increased numbers of Dlb-1 mutations. Mutation induction in Ahr-non-responsive mice in the absence of detectable CYP1A1 in either liver or small intestine indicates that an appreciable portion of B[a]P activation to a genotoxin must be by other than a CYP1A1 mediated route. These data show that B[a]P is a potent small intestinal mutagen at the Dlb-1 locus.