The gene expression of liver major gap junction (GJ) protein was studied in rats systemically administered phenobarbital, a rat liver tumor promoter. Using a GJ protein cDNA and northern blot analysis, the level of GJ protein mRNA in liver was observed to be markedly reduced at 4 and 11 wk of phenobarbital exposure (0.1% in drinking water). However, the level of GJ protein mRNA was not altered in kidney at 11 wk of exposure. In liver, phenobarbital did not induce expression of the neoplasm-associated marker genes glutathione S-transferase (placental form) and gamma-glutamyltranspeptidase, while in kidney the observed expression of these genes was not changed. These in vivo results indicate that phenobarbital reduces GJ protein gene expression specifically in rat liver without altering expression of genes often altered during liver carcinogenesis, and they support assigning a role for the impairment of gap junctional intercellular communication in phenobarbital-mediated liver tumor promotion.