Insulin inhibits the hepatic transcription of insulin-like growth factor binding protein-1 (IGFBP-1). In the present studies, human HEP G2 hepatoma cells were transiently transfected with human IGFBP-1 gene promoter constructs in order to identify cis elements and trans-acting factors that confer the insulin effect. Transfections of IGFBP-1 promoter deletion constructs localized an insulin responsive element (IRE) between approximately 140- and approximately 103-base pair (bp) 5' to the mRNA capsite. This region contains a 25-bp sequence which is 100% conserved in the rat IGFBP-1 promoter and which has two AT-rich, 8-bp elements exhibiting dyad symmetry. Site-directed mutagenesis of both elements in the same 1205-bp IGFBP-1 promoter construct abolished the inhibitory effect of insulin on promoter activity. Also, the native but not the mutant IGFBP-1 IRE conferred the inhibitory effect of insulin to the heterologous thymidine kinase promoter. Gel mobility shift assays identified a DNA binding activity which specifically binds the native IGFBP-1 IRE and which is not altered by prior insulin treatment. The IGFBP-1 IRE sequence is similar to those of functionally mapped IREs from other gene promoters, suggesting that this common IRE and the protein(s) which it binds confer the insulin effect to a number of insulin-sensitive genes.