Areca quid (AQ) chewing and smoking have synergistic potential in the development of oral squamous cell carcinoma (OSCC). In Taiwan, fresh Piper betle inflorescence is uniquely added to AQ, and hydroxychavicol (HC) is the major phenolic component of P. betle inflorescence. This study investigated whether HC modulates cigarette carcinogen benzo[a]pyrene (B[a]P)-mediated toxic effects. Pretreatment of HC and followed by B[a]P challenge resulted in higher cytotoxicity and HPRT gene mutation frequency (P < 0.05). However, this treatment protocol resulted in decreased bulky B[a]P-DNA adduct levels as demonstrated by 32P-postlabeling technique (P < 0.05). Western blotting analysis indicated that HC pretreatment induced the expression of cyclooxygenase-2 (COX-2) and dihydrodiol dehydrogenase (DDH). COX-2 is know to participate in the B[a]P-DNA adduct formation, while DDH has been shown to divert B[a]P-diol to B[a]P-7,8-quinone and the generation of reactive oxygen species (ROS). Using flow cytometry, this study demonstrated the increased production of 8-oxoguanine (P < 0.001). Overall, the results suggest that HC-induced DDH is more important than site-by-site up-regulation of COX-2 in B[a]P-induced cytotoxicity and HPRT gene mutation. Furthermore, DDH-mediated oxidative DNA damage and not B[a]P-DNA adduct formation may be involved in the HC and B[a]P-induced toxic effects.