The DNA replication-related element-binding factor (DREF) regulates cell proliferation-related gene expression in Drosophila. We have carried out a genetic screening, taking advantage of the rough eye phenotype of transgenic flies that express full-length DREF in the eye imaginal discs and identified the eukaryotic initiation factor 4A (eIF4A) gene as a dominant suppressor of the DREF-induced rough eye phenotype. The eIF4A gene was here found to carry three DRE sequences, DRE1 (-40 to -47), DRE2 (-48 to -55), and DRE3 (-267 to -274) in its promoter region, these all being important for the eIF4A gene promoter activity in cultured Drosophila Kc cells and in living flies. Knockdown of DREF in Drosophila S2 cells decreased the eIF4A mRNA level and the eIF4A gene promoter activity. Furthermore, specific binding of DREF to genomic regions containing DRE sequences was demonstrated by chromatin immunoprecipitation assays using anti-DREF antibodies. Band mobility shift assays using Kc cell nuclear extracts revealed that DREF could bind to DRE1 and DRE3 sequences in the eIF4A gene promoter in vitro, but not to the DRE2 sequence. The results suggest that the eIF4A gene is under the control of the DREF pathway and DREF is therefore involved in the regulation of protein synthesis.