The effects of pH and temperature on benzo[a]pyrene(BaP)-DNA, -protein, and -lipid adducts were compared in rat hepatocytes treated with [3H]-BaP. In primary rat hepatocytes, BaP-DNA, -protein, and -lipid adducts were formed in a concentration-dependent manner (0.31-2.80 mmol BaP), and the formation of BaP-lipid adducts, specifically triglyceride (TG) adducts, correlated well with that of DNA or protein adducts. As incubation time increased (1-24 h), the formation of DNA or protein adducts was also elevated, while BaP-TG adducts decreased after 3 h of reaction. pH had no effect on the formation of [3H]-BaP-DNA, and -protein adducts, and the TG adducts decreased with increasing pH (3.4-11.4). With an increase in incubation temperature (30-45 degrees C), on the other hand, the adduct formation was increased with increasing temperature up to 40 degrees C and then decreased sharply. These results suggest that the physicochemical properties of BaP-DNA, -protein, and -TG adducts might be important to consider when cells or humans who have been exposed to BaP are monitored for biomarkers.