Primary cultures of rat hepatocytes were exposed to [ring-3H]-N-hydroxy-2-acetylaminofluorene (N-OH-AAF) for 4 h, and the RNA and DNA nucleoside adducts were isolated and identified by h.p.l.c. The DNA adducts were shown to be N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-C8-AAF), N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-C8-AF), and 3-(deoxy-guanosin-8-yl)-2-acetylaminofluorene (dG-N2-AAF), while the RNA adducts were N-(guanosin-8-yl)-2-acetyl-aminofluorene, and N-(guanosin-8-yl)-2-aminofluorene. The removal of these adducts was measured up to 38 h following the cessation of exposure of the hepatocytes to N-OH-AAF. The dG-C8-AAF adduct was removed with a half-life of approximately 10 h, while dG-N2-AAF and dG-C8-AF remained constant for 14 h, followed by a slow rate of removal. The dG-C8-AAF adduct initially composed about 60%of the total DNA adducts of primary hepatocytes in contrast to the 20%found in liverin vivo. The formation of the 3 DNA adducts and the different rates of repair indicate that primary cultured rat hepatocytes may be a valuable system to study initiation of liver carcinogenesis by N-OH-AAF.