Indole-3-carbinol (I3C) and phenobarbital (PB) are cytochrome P450 (CYP) 1A and CYP2B inducers, respectively, and have liver tumor-promoting effects in rats. In this study, we investigated the modifying effects on tumor promotion by I3C and PB co-administration. Six-week-old male F344 rats received a single intraperitoneal injection of N-diethylnitrosamine for initiation treatment. Two weeks after the initiation, rats were given no tumor-promoting agents (DEN alone), I3C (2,500 or 5,000 ppm in diet), PB (60 or 120 ppm in drinking water), or 2,500 ppm I3C + 60 ppm PB for 6 weeks. One week after the I3C/PB treatments, all animals underwent a two-thirds partial hepatectomy. The number and area of liver cell foci positive for glutathione S-transferase placental form (GST-P(+) foci) were not significantly fluctuated in the PB+I3C group in the isoadditive statistical model. On the contrary, the mRNA levels of Cyp2b1/2 and Nqo1 were suppressed and enhanced, respectively, in the PB+I3C group in the isoadditive model, but there was no enhancement in the microsomal reactive oxygen species (ROS) production, thiobarbituric acid-reactive substance levels, and Ki-67(+) cell ratio in this group. The results suggest that the co-administration of I3C and PB causes no modifying effects in liver tumor promotion in rats.