In search of genes that are steadily up-regulated during the promotion stage in carcinogenesis, suppression PCR subtractive hybridization and following northern blot screening were performed using a phenobarbital (PB)-promotion model based on a medium-term liver bioassay. Two weeks after a single injection of diethylnitrosamine (DEN; 200 mg/kg body wt, i.p.), rats were given 600 p.p.m. PB in the drinking water for up to 64 weeks. For comparison, animals fed 1 p.p.m. ethinylestradiol (EE) or 3000 p.p.m. butylated hydroxytoluene (BHT) in the diet at promotion stage were also included. Rats were subjected to partial hepatectomy (PH) at week 3. In addition, dose-dependence of PB at week 8 of promotion and responsiveness to representative non-genotoxic carcinogens without DEN initiation were examined. Fragments of a total of 67 different genes were isolated from the up-regulated gene population in the liver at day 10 of PB treatment by subtracting from basal expression of DEN + PH alone. Using northern blot screening for signal-detectable 48 genes, 16 genes showed up-regulation in the livers at week 8 of promotion, common to the PB and EE treatments with the levels being three times or more than the basal expression of unpromoted liver. The majority of these genes were also up-regulated at week 8 by BHT treatment, and were also constitutively expressed in the DEN(-), PH(-) untreated rat livers. Among the up-regulated genes common to the PB and EE promotion, and not responding to the non-genotoxic carcinogens in uninitiated liver, the following six genes showed overexpression in PB-promoted hepatocellular carcinomas at week 64, with the levels three times or more than untreated rat liver: ubiquitously expressed mammalian ABC half transporter, apolipoprotein A4, nuclear receptor binding factor-2, CD81, hypothetical protein (HSPC014) and one unidentified gene. These genes might be candidates for biomarkers in screening of non-genotoxic hepatocarcinogens by analysis in two-stage carcinogenesis models.