Glucosinolates are common components of cruciferous vegetables that can be hydrolyzed during food processing to yield isothiocyanates, some of which have been shown to inhibit the induction of mammary tumors in rats by 7,12-dimethyl-benz[a]anthracene. To determine how intact glucosinolates affect the metabolism of benzo[a]pyrene (B[a]P) in mammalian cells in culture, the effects of a series of glucosinolates on the metabolism and DNA binding of B[a]P were investigated in early passage Syrian hamster embryo cell cultures. Glucolimnanthin, a glucosinolate from Limnanthes douglasii increased the amount of B[a]P metabolized by the hamster embryo cell cultures during a 24 h exposure. The glucolimnanthin-treated cultures contained a higher proportion of B[a]P-phenol glucuronides and other water-soluble metabolites than control cultures. Cotreatment with glucolimnanthan and [3H]B[a]P for 24 h resulted in a greater than 2-fold increase in the amount of B[a]P bound to DNA and a 3-fold increase in the amount of deoxyguanosine adduct formed by reaction of 7R,8S-dihydroxy-9S,10R-epoxy-7,8,9,10-tetrahydroB[a]P [(+)-anti-B[a]PDE]. The glucolimnanthin was metabolized essentially completely within 24 h. An increase in B[a]P metabolism similar to that caused by glucolimnanthin was induced by cotreatment of hamster embryo cell cultures with m-methoxybenzyl isothiocyanate, a metabolite that can be formed from glucolimnanthin by enzymatic hydrolysis. These results indicate that the glucosinolate glucolimnanthin can increase the metabolic activation of B[a]P in mammalian cells in culture.