The rat placental glutathione S-transferase (GST-P), an isozyme of glutathione S-transferase (GST), is not expressed in normal liver, but is highly induced at an early stage of chemical hepatocarcinogenesis and in hepatomas. Recently, we reported that the Nrf2/MafK heterodimer binds to GPE1, a strong enhancer of the GST-P gene, and activates this gene in pre-neoplastic lesions and hepatomas. In addition to the positive regulation during hepatocarcinogenesis, negative regulatory mechanisms might work to repress GST-P in normal liver, but this remains to be clarified. In this work, we identified the CCAAT enhancer binding protein (C/EBP as a negative regulator that binds to GPE1 and suppresses GST-P expression in normal liver. C/EBP binds to the part of GPE1 sequence and that the binding of Nrf2/MafK and C/EBP to GPE1 occurs in a mutually exclusive. In a transient-transfection analysis, C/EBP activated GPE1 in F9 embryonal carcinoma cells, but strongly inhibited GPE1 activity in hepatoma cells. The expression of C/EBP was specifically suppressed in GST-P-positive pre-neoplastic foci in the livers of carcinogen-treated rats. A chromatin immunoprecipitation analysis showed that C/EBP bound to GPE1 in the normal liver in vivo, but did not bind in pre-neoplastic hepatocytes. Introduction of the C/EBP gene fused with the estrogen receptor ligand-binding domain into hepatoma cells and subsequent activation by -estradiol led to suppression of endogenous GST-P expression. These results indicate that C/EBP is a negative regulator of GST-P gene expression in normal liver.