Rat GH gene expression is known to be stimulated by several factors, including thyroid hormone and GRF. This effect of GRF appears to be mediated by cAMP resulting from activation of adenylate cyclase by the peptide. The elements of the rat GH gene important for thyroid hormone stimulation and cell-specific expression have been previously mapped using gene transfection techniques. Cell-specific expression of the gene is mediated by two cell-specific elements located from −137 to −107 and from −95 to −65. Sequences mediating thyroid hormone stimulation are thought to be located between −208 and −160. In this study, using three different methods to elevate cAMP levels in cells [forskolin, a direct activator of the adenylate cyclase catalytic subunit; 8-(4-chlorophenylthio)cAMP, a nonmetabolizable cAMP analog; and isobutylmethylxanthine, a phosphodiesterase inhibitor], we show that 5′-flanking DNA of the rat GH gene can mediate stimulation by cAMP (10- to 20-fold). The cAMP-responsive region was mapped to sequences between −104 and +11, which contains the proximal cell-specific element (−95/−65) important for cell-specific expression. Either the −97/ −65 or the −104/−47 region of the gene, cloned upstream of a heterologous promoter, conferred only minimal or no activation by cAMP. This suggests that these sequences are not the direct target of cAMP action or that they are insufficient alone to mediate the cAMP response. The cAMP regulatory element (TGACGTCA) is not found between −104 and +11, and cAMP activation does not appear to act via putative AP-2 elements, since phorbol esters did not stimulate expression. In gene constructs containing sequences that mediate thyroid hormone stimulation (extending to −208), cAMP effectors and l-T₃ act synergistically, suggesting a cooperative effect of these distinct cis-acting regions to activate rat GH gene expression.