Using the c-fosenhancer as a model to analyze growth hormone (GH)-promoted gene expression, the roles of CCAAT/enhancer-binding proteins (C/EBPs) in GH-regulated transcription were investigated. In 3T3-F442A fibroblasts stably expressing the c-fospromoter mutated at the C/EBP binding site upstream of luciferase, c-fospromoter activity is stimulated by GH 6–7-fold; wild type c-fospromoter shows only a 2-fold induction by GH. This suggests that C/EBP restrains GH-stimulated expression of c-fos. Electrophoretic mobility shift assays with nuclear extracts from 3T3-F442A cells indicate that GH rapidly (2–5 min) increases binding of C/EBPβ and C/EBPδ, to the c-fosC/EBP binding site. Both liver activating protein (LAP) and liver inhibitory protein (LIP), forms of C/EBPβ, are detected in 3T3-F442A cells by immunoblotting. GH increases the binding of LAP/LAP and LAP/LIP dimers. Overexpression of LIP interferes with GH-promoted reporter expression in CHO cells expressing GH receptors, consistent with the possibility that LIP restrains GH-stimulated c-fosexpression. Overexpression of LAP elevates basal luciferase activity but does not influence promoter activation by GH, while overexpressed C/EBPδ elevates basal promoter activity and enhances the stimulation by GH. GH stimulates the expression of mRNA for C/EBPβ and -δ and increases levels of C/EBPδ. Although C/EBPβ is not detectably altered, GH induces a shift to more rapidly migrating forms of LIP and LAP upon immunoblotting. Treatment of extracts from GH-treated cells with alkaline phosphatase causes a shift of the slower migrating form to the rapidly migrating form, consistent with GH promoting dephosphorylation of LIP and LAP. These studies implicate C/EBPβ and -δ in GH-regulated gene expression. They also indicate that GH stimulates the binding of C/EBPβ and -δ to the c-fospromoter and promotes the dephosphorylation of LIP and LAP. These events may contribute to the ability of C/EBPβ and -δ to regulate GH-stimulated expression of c-fos.