The mouse lactoferrin gene responded to forskolin, 12-O-tetradecanoyl phorbol-13-acetate, and epidermal growth factor (EGF) stimulation via two adjacent enhancer elements, the cAMP response element (CRE) and EGF response element (EGFRE), collectively referred to as the mitogen response unit. In this report, we examined the minimal promoter and enhancer elements of the mouse lactoferrin gene that are required for EGF-induced transcriptional activation. We found that the CRE and noncanonical TATA box (ATAAA) are the minimal promoter elements for basal activity of the chloramphenicol acetyltransferase (CAT) reporter construct whereas the EGFRE is needed for an additional activity induced by EGF in transiently transfected human endometrial carcinoma RL95-2 cells (RL95-2). The EGFRE, however, did not function in heterologous promoters [SV 40 and thymidine kinase (TK)]. Therefore, EGF-stimulated lactoferrin gene activity is promoter specific in RL95-2 cells. In transiently transfected cells, EGF and forskolin showed synergistic effects on the CAT reporter that contained both response elements. Mutation made at either element or insertion of extra nucleotides between the two elements severely affected EGF-stimulated activity. Nuclear protein prepared from RL95-2 cells formed three complexes (A, B, and C) with the oligonucleotides containing both EGFRE and CRE in electrophoretic mobility shift assay. A new complex (E) was detected with the nuclear protein of EGF-treated cells. By oligonucleotide competition experiments, we demonstrated that the complex E was generated by protein bound to CRE. EGF-induced binding activity could be abolished by calf intestinal alkaline phosphatase but not by the protein synthesis inhibitor, cycloheximide. Therefore, binding of a preexisting phosphoprotein to the CRE region could be one of the requirements for EGF-induced mouse lactoferrin gene promoter activity.