Expression of the human insulin-like growth factor I (hIGF-I) gene is regulated in a tissue- and developmental stage-specific manner. The hIGF-I gene has two promoters, P1 and P2. P1 is the more active promoter by far in adult liver, the main endocrine source of IGF-I. Recently, we described the involvement of the CAAT/enhancer binding protein family of liver-enriched transcription factors in the regulation of the expression of IGF-I in adult liver. In this study we report on the role of another family of liver-enriched transcription factors in the regulation of IGF-I expression. Hepatocyte nuclear factor 1 alpha (HNF-1 alpha) is shown to transactivate IGF-I P1 in transient transfection experiments performed in Hep3B cells. Bandshift experiments reveal that two distinct regions in P1, located 119 and 282 nucleotides upstream of the transcription start site, can bind HNF-1 alpha with relatively high affinity. Both HNF-1-binding sites are evolutionary well conserved, emphasizing the importance of HNF-1 in the regulation of the IGF-I gene expression. Mutational analysis of the binding sites indicates that both sites are essential for maximal stimulation of P1 by HNF-1 alpha, although the largest contribution stems from the more downstream of the two HNF-1-binding sites. The latter site completely overlaps the previously described CAAT/enhancer binding protein binding site in P1. The colocalization of the binding sites, to which binding of the respective factors seems to be mutually exclusive, is suggestive of a regulatory hotspot to which members of different transcription factor families may bind depending on developmental stage and nutritional status.