It has been speculated that oxidative stress is involved in the liver tumor-promoting effect of β-naphthoflavone (BNF; 5,6-benzoflavone) in rats, because of its strong induction of cytochrome P450 (CYP) 1A enzymes. In order to clarify the mechanism of liver tumor promoting effects of BNF, male F344 rats were initiated with a single intraperitoneal injection of 200 mg/kg diethylnitrosamine (DEN), and fed diet containing 2% of BNF for 6 weeks staring 2 weeks after injection. Two/third partial hepatectomy was perfomed at Week 3 after the treatment of BNF. Low-density Rat Toxicology & Drug Resistance Microarray and quantitative analyses of mRNA expressions of the selected genes using real-time reverse transcription (RT) -PCR were carried out on total RNAs extracted from the livers of F344 rats. Collected liver tissues were subjected to light microscopic examinations (hematoxylin and eosin staining), immunohistochemistries of proliferating cell nuclear antigen (PCNA), glutathione S-transferase placental form (GST-P) and CYP1A1, and Schmorl staining to identify lipofuscin. Gene expression analysis showed that 7 genes (CYP1A1, CYP1A2, CYP1B1, Gstm2 (GST mu2), Gstm3, glutathione peroxidase (Gpx)2 and NAD(P)H dehydrogenase, quinone 1(Nqo1)) were up-regulated (> 1.5 fold), and 4 genes (8-oxoguanine DNA glycosylase (Ogg1), Gpx1, peroxiredoxin (Prdx) 1 and P450 oxidoreductase (Por)) were down-regulated (< 0.67 fold) in the DEN + BNF group rats as compared with the DEN alone group. In immunohistochemical analyses, the numbers of foci positive for GST-P, the rate of PCNA-positive cells, and the rate of CYP1A1-positive cells were significantly increased in the DEN + BNF group, as compared to the corresponding control. Furthermore, deposition of lipofuscin, positive for Schmorl staining, was moderately increased in the DEN + BNF group. These results confirm the possibility that oxidative stress is involved in the liver tumor-promoting effect of BNF in rats.