In hepatocytes, insulin-like growth factor-binding protein-1 (IGFBP-1) levels are increased by glucocorticoids and by agents that raise intracellular cAMP levels such as glucagon, theophylline, forskolin, and cAMP analogues. In contrast, insulin lowers IGFBP-1 levels, an effect dominant over the glucocorticoid and cAMP effects. Previous studies showed that dibutyryl cAMP (Bt2cAMP) and theophylline increase IGFBP-1 promoter activity in HEP G2 human hepatoma cells and that insulin abolishes this increase. In studies reported here, HEP G2 cells were used to further evaluate the role of cAMP in stimulating IGFBP-1 expression. Initial studies found that either 0.5 or 5.0 mM Bt2cAMP alone, or the combination of 0.5 mM Bt2cAMP and 2 mM theophylline, increased IGFBP-1 protein levels, mRNA levels, and promoter activity, but that the addition of theophylline to Bt2cAMP was required to give a approximately 5-fold increase in promoter activity. Deletion mutations of the IGFBP-1 promoter were used to show that much of the effect of Bt2cAMP and theophylline was conferred by the region between 269 and 246 base pairs (bp) 5' of the IGFBP-1 mRNA cap site. DNase I protection assays showed that HEP G2 nuclear extract footprinted the region from 273 to 249 bp 5' of the cap site; this region, designated P2, has a central CGTCA motif common to cAMP-responsive elements (CREs). Mutating the CGTCA motif in the 1205-bp IGFBP-1 promoter construct to TAGCA led to a 51% decrease in the ability of Bt2cAMP and theophylline to stimulate IGFBP-1 promoter activity above control levels. In addition, cotransfection of the catalytic subunit of cAMP-dependent protein kinase A (PKA) with the native 1205-bp IGFBP-1 promoter construct stimulated IGFBP-1 promoter activity 3.9-fold, but the TAGCA mutation decreased by 73% the ability of PKA to stimulate IGFBP-1 promoter activity above control levels. Mutating the CGTCA motif to TAGCA also blocked the ability of both crude HEP G2 nuclear extract and recombinant CRE-binding protein to bind to the P2 element. These data suggest that the P2 element is a CRE that confers at least part of the stimulatory effect of cAMP on the human IGFBP-1 promoter.