gamma-Glutamyl transpeptidase (GGT) is an enzyme encoded by multiple mRNAs (mRNAI to mRNAIV) that, in the rat, are transcribed from a single copy gene in a tissue-specific manner. In the liver, GGT expression is up-regulated in transformed cells, and this induction is the most widely used marker of liver cell transformation. We characterized the GGT mRNA species expressed in the liver (mRNAIII), and we report that this mRNA differs from the other GGT mRNA species by a 275-base alternate 5'-end sequence. Its transcription occurs on a specific promoter (promoter III) that maps on the GGT gene upstream of the two promoters coding for the GGT mRNAI and mRNAII. In hepatoma cells, mRNAIII expression is related to the differentiation state of the cells. We have shown that, in Reuber H-35-derived cell lines, the GGT mRNAIII is transcribed in cells that express a differentiated phenotype (Fao), but not in the dedifferentiated C2 and H5 variants. Moreover, we observed a reexpression of the GGT mRNAIII species in the C2 Rev7 variant, which has reverted from C2 toward a differentiated hepatocyte phenotype. In the proximal promoter III region, we identified a sequence that strongly enhances transcriptional activity in Fao and C2 Rev7 cells, but not in the dedifferentiated C2 variant. This motif interacts with nuclear proteins belonging to the NF-1 and NF-Y families that govern GGT promoter III expression in differentiated hepatoma cells.