The human gene coding for insulin-like growth factor II (IGF-II) contains four promoters (P1-P4), that are subjected to tissue-specific and development-dependent regulation. Expression of promoter P1 is detected only in adult liver tissue, whereas promoter P3 is the major IGF-II promoter in fetal liver and is further expressed in other fetal tissues and in adult non-hepatic tissues. C/EBP is a tissue- and development-specific transcription factor that is expressed predominantly in adult liver, adipose tissue and lung. The effect of C/EBP on the expression of constructs containing IGF-II promoter P1 or P3 linked to the luciferase gene was investigated in cotransfection assays using Hep3B cells. We found that promoter P1 can be activated by C/EBP, whereas this transcription factor has no effect on the expression of promoter P3. By gel retardation and DNasel footprinting it was demonstrated that C/EBP can bind to a region of P1 located between 82 and 109 basepairs upstream of the cap site. Furthermore, we showed that deletion of this C/EBP binding region strongly reduces the ability of C/EBP to stimulate transcription from P1. These results indicate that C/EBP is a major component in the specific activation of the human IGF-II promoter P1 in adult liver.