The human insulin-like growth factor II (IGF-II) gene contains four promoters, P1-P4. In fetal liver promoter P3 is the major promoter, which consists of a proximal region that supports general transcription, and a cell-specific region located more upstream. In addition to the TATA box, the proximal region contains four binding sites for nuclear proteins, designated PE3-1 to PE3-4. To determine the influence of the proteins binding to these elements, the transcriptional activity of the proximal region of P3 was investigated. Promoter P3 was analyzed in Hep3B cells, which express high levels of IGF-II mRNA derived from this promoter, and in HeLa cells, that have an inactive IGF-II gene. By analysis of 5'-deletion constructs in an in vitro transcription system and in transient expression assays, and by competition with specific oligonucleotides it was shown that the factors binding to the elements PE3-4, PE3-2 and PE3-1 play an important role in the regulation of promoter P3.