The study was aimed at determining the role of glutathione (GSH) conjugation in the binding of reactive benzo[a]pyrene (BaP) species to DNA of C3H/10T1/2 cells. In order to suppress GSH conjugation cells were depleted of GSH by treatment with buthionine sulfoximine for 18 h and 1-chloro-2,4-dinitrobenzene for 1 h prior to incubation with radiolabelled substrates. Under these conditions GSH levels decreased to less than 1% of the control value. C3H/10T1/2 cells produced GSH conjugates with 7,8-dihydroxy-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BaPDE) comprising 6% of the total metabolites formed from BaP or (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (BaP-7,8-diol). In GSH-depleted cells formation of GSH conjugates with metabolic products of BaP or BaP-7,8-diol was suppressed to 1% of total metabolites during an 8-h incubation period. Metabolic activation of BaP and BaP-7,8-diol by C3H/10T1/2 cells resulted in the formation of DNA adducts which largely consisted of BaPDE:deoxyguanosine. Depletion of GSH altered neither the degree of DNA binding nor the pattern of DNA adducts to any significant extent. When C3H/10T1/2 cells were co-incubated with microsomes from liver of 3-methylcholanthrene-treated rats for 1 h in order to activate BaP or BaP-7,8-diol extracellularly, the same pattern of GSH conjugates and DNA adducts was generated as by intracellular metabolism of the polycyclic hydrocarbons. No GSH conjugates were detected following co-incubation of microsomes with GSH-depleted C3H/10T1/2 cells. The formation of DNA adducts again remained unaffected by the suppression of conjugation. C3H/10T1/2 cells are apparently capable of conjugating BaPDE with GSH but are not capable of trapping by GSH conjugation those BaPDE moieties which bind to DNA. The results are compatible with the notion that BaPDE is partially contained in a cellular compartment--presumably the lipid environment of membranes--where it is inaccessible to GSH transferases of C3H/10T1/2 cells.