Insulin-like growth factor-binding protein-1 (IGFBP-1) modulates the action of IGFs on target cells. IGFBP-1 transcription is highly regulated by hormonal and metabolic factors. In rat H4-II-E hepatoma cells, IGFBP-1 messenger RNA is stimulated by dexamethasone, cAMP, and phorbol esters, and dominantly inhibited by insulin. To identify the cis-elements that determine transcriptional regulation by these agents, we have coupled rat IGFBP-1 promoter fragments to a luciferase reporter gene and transfected H4-II-E cells using the cationic liposome procedure. Promoter fragments whose 5'-end was at nucleotide (nt) -925 or -327 (with respect to the transcription initiation site, 1) conferred positive regulation of promoter activity by dexamethasone, cAMP, and phorbol esters. Insulin inhibited promoter activity in the presence of any of the three stimulatory agents. Stimulation by cAMP or phorbol esters was abolished when the region between nt -327 and -235 was deleted. Although this region contains potential activating protein-2 and activating protein-1 sites, the sites responsible for this regulation have not yet been identified. By contrast, stimulation by dexamethasone was retained in deletion constructs whose 5'-end was at nt -92, but was abolished by site mutagenesis of either the left or right half-sites of a potential glucocorticoid response element (GRE) located between nt -91 and -77. Surprisingly, substitution mutations in an up-stream region, -108 to -99 (M4), also decreased dexamethasone-stimulated promoter activity despite the presence of an intact GRE. We postulate that a positive factor that binds to the wild-type M4 region neutralizes factors that inhibit interaction of the glucocorticoid receptor with the GRE. The M4 region also is involved in inhibition by insulin. Insulin inhibition of dexamethasone-stimulated promoter activity was lost after deletion of nt -135 to -92 or mutation of the region between nt -108 and -99. This insulin response element is conserved in the human IGFBP-1 promoter and is homologous to the insulin response element of the phosphoenolpyruvate carboxykinase gene, which also is rapidly inhibited by insulin in H4-II-E cells. The rat IGFBP-1 promoter provides a valuable model system for studying the multihormonal regulation of transcription.