GH regulates gene expression by modulating the concentration or activity of transcription factors. To identify transcription factors that mediate the effects of GH in liver we analyzed the promoter of the gene coding for hepatocyte nuclear factor-6 (HNF-6), whose expression in liver is stimulated by GH. In protein-DNA interaction studies and in transfection experiments, we found that the liver-enriched transcription factor CCAAT/enhancer-binding protein-alpha (C/EBPalpha) binds to the hnf6 gene and inhibits its expression. This inhibitory effect involved an N-terminal subdomain of C/EBPalpha and two sites in the hnf6 gene promoter. Using liver nuclear extracts from GH-treated hypophysectomized rats, we found that GH induces a rapid, transient decrease in the amount of C/EBPalpha protein. This GH-induced change is concomitant with the transient stimulatory effect of GH on the hnf6 gene. Stimulation of the hnf6 gene by GH therefore involves lifting of the repression exerted by C/EBPalpha in addition to the known GH-induced stimulatory effects of STAT5 (signal transducer and activator of transcription-5) and HNF-4 on that gene. Our data provide further evidence that GH controls a network of liver transcription factors and show that C/EBPalpha participates in this process.