The CCAAT/enhancer binding protein delta (C/EBPdelta, CRP3, CELF, NF-IL6beta) regulates gene expression and plays functional roles in many tissues, such as in acute phase response to inflammatory stimuli, adipocyte differentiation, and mammary epithelial cell growth control. In this study, we examined the expression of human C/EBPdelta (NF-IL6beta) gene by epidermal growth factor (EGF) stimulation in human epidermoid carcinoma A431 cells. NF-IL6beta was an immediate-early gene activated by the EGF-induced signaling pathways in cells. By using 5'-serial deletion reporter analysis, we showed that the region comprising the -347 to +9 base pairs was required for EGF response of the NF-IL6beta promoter. This region contains putative consensus binding sequences of Sp1 and cAMP response element-binding protein (CREB). The NF-IL6beta promoter activity induced by EGF was abolished by mutating the sequence of cAMP response element or Sp1 sites in the -347/+9 base pairs region. Both in vitro and in vivo DNA binding assay revealed that the CREB binding activity was low in EGF-starved cells, whereas it was induced within 30 min after EGF treatment of A431 cells. However, no change in Sp1 binding activity was found by EGF treatment. Moreover, the phosphatidylinositol 3 (PI3)-kinase inhibitor (wortmannin) and p38(MAPK) inhibitor (SB203580) inhibited the EGF-induced CREB phosphorylation and the expression of NF-IL6beta gene in cells. We also demonstrated that CREB was involved in regulating the NF-IL6beta gene transcriptional activity mediated by p38(MAPK). Our results suggested that PI3-kinase/p38(MAPK)/CREB pathway contributed to the EGF activation of NF-IL6beta gene expression.