The aim of this study was to generate and identify a novel benzo[a]pyrene (BP)-derived DNA adduct found both in vitro and in vivo, To date, the majority of studies have focused on N-2-[10 beta (7 beta ,8a,9a-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene)yl]-deoxyguanosine (anti-BPDE-dG), the major adduct generated following bioactivation of BP, However, a second adduct is also formed following bioactivation of BP which has been speculated to result from further metabolism of 9-OH-BP, In order to identify this second reaction pathway, the ultimate DNA binding species, and the DNA base involved, we have synthesized and characterized a dG-derived DNA adduct arising from further bioactivation of 9-OH-BP in the presence of rat liver microsomes, Analysis of the adducted nucleotides was conducted using both the P-32-postlabeling assay and capillary electrophoresis-mass spectrometry (CE-MS),